Mechanisms of Action of trans Fatty Acids
Oteng, Antwi Boasiako ; Kersten, Sander - \ 2020
Advances in Nutrition 11 (2020)3. - ISSN 2161-8313 - p. 697 - 708.
cardiometabolic disease - cholesterogenesis - elaidic acid - ER stress - industrial trans fatty acid - inflammation - lipid metabolism - ruminant trans fatty acid
Human studies have established a positive association between the intake of industrial trans fatty acids and the development of cardiovascular diseases, leading several countries to enact laws that restrict the presence of industrial trans fatty acids in food products. However, trans fatty acids cannot be completely eliminated from the human diet since they are also naturally present in meat and dairy products of ruminant animals. Moreover, bans on industrial trans fatty acids have not yet been instituted in all countries. The epidemiological evidence against trans fatty acids by far overshadows mechanistic insights that may explain how trans fatty acids achieve their damaging effects. This review focuses on the mechanisms that underlie the deleterious effects of trans fatty acids by juxtaposing effects of trans fatty acids against those of cis-unsaturated fatty acids and saturated fatty acids (SFAs). This review also carefully explores the argument that ruminant trans fatty acids have differential effects from industrial trans fatty acids. Overall, in vivo and in vitro studies demonstrate that industrial trans fatty acids promote inflammation and endoplasmic reticulum (ER) stress, although to a lesser degree than SFAs, whereas cis-unsaturated fatty acids are protective against ER stress and inflammation. Additionally, industrial trans fatty acids promote fat storage in the liver at the expense of adipose tissue compared with cis-unsaturated fatty acids and SFAs. In cultured hepatocytes and adipocytes, industrial trans fatty acids, but not cis-unsaturated fatty acids or SFAs, stimulate the cholesterol synthesis pathway by activating sterol regulatory element binding protein (SREBP) 2-mediated gene regulation. Interestingly, although industrial and ruminant trans fatty acids show similar effects on human plasma lipoproteins, in preclinical models, only industrial trans fatty acids promote inflammation, ER stress, and cholesterol synthesis. Overall, clearer insight into the molecular mechanisms of action of trans fatty acids may create new therapeutic windows for the treatment of diseases characterized by disrupted lipid metabolism.
Akkermansia muciniphila Exerts Lipid-Lowering and Immunomodulatory Effects without Affecting Neointima Formation in Hyperlipidemic APOE*3-Leiden.CETP Mice
Katiraei, Saeed ; Vries, Margreet R. de; Costain, Alice H. ; Thiem, Kathrin ; Hoving, Lisa R. ; Diepen, Janna A. van; Smits, Hermelijn H. ; Bouter, Kristien E. ; Rensen, Patrick C.N. ; Quax, Paul H.A. ; Nieuwdorp, Max ; Netea, Mihai G. ; Vos, Willem M. de; Cani, Patrice D. ; Belzer, Clara ; Dijk, Ko Willems van; Berbée, Jimmy F.P. ; Harmelen, Vanessa van - \ 2019
Molecular Nutrition & Food Research (2019). - ISSN 1613-4125
Akkermansia muciniphila - atherosclerosis - immunity - lipid metabolism - mesenteric lymph nodes
Scope: Akkermansia muciniphila (A. muciniphila) is an intestinal commensal with anti-inflammatory properties both in the intestine and other organs. The aim is to investigate the effects of oral administration of A. muciniphila on lipid metabolism, immunity, and cuff-induced neointima formation in hyperlipidemic APOE*3-Leiden (E3L).CETP mice. Methods and results: Hyperlipidemic male E3L.CETP mice are daily treated with 2 × 108 CFU A. muciniphila by oral gavage for 4 weeks and the effects are determined on plasma lipid levels, immune parameters, and cuff-induced neointima formation and composition. A. muciniphila administration lowers body weight and plasma total cholesterol and triglycerides levels. A. muciniphila influences the immune cell composition in mesenteric lymph nodes, as evident from an increased total B cell population, while reducing the total T cell and neutrophil populations. Importantly, A. muciniphila reduces the expression of the activation markers MHCII on dendritic cells and CD86 on B cells. A. muciniphila also increases whole blood ex vivo lipopolysaccharide-stimulated IL-10 release. Finally, although treatment with A. muciniphila improves lipid metabolism and immunity, it does not affect neointima formation or composition. Conclusions: Four weeks of treatment with A. muciniphila exerts lipid-lowering and immunomodulatory effects, which are insufficient to inhibit neointima formation in hyperlipidemic E3L.CETP mice.
Unravelling mechanisms of dietary flavonoid-mediated health effects: effects on lipid metabolism and genotoxicity
Hoek-van den Hil, E.F. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens; Jaap Keijer, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462573031 - 157
flavanoïden - flavonoïden - vetzuren - quercetine - flavonolen - lichaamsgewicht - lipidenmetabolisme - hart- en vaatziekten - lever - vetweefsel - gezondheid - genotoxiciteit - voeding - muizen - flavanoids - flavonoids - fatty acids - quercetin - flavonols - body weight - lipid metabolism - cardiovascular diseases - liver - adipose tissue - health - genotoxicity - nutrition - mice
Consumption of foods containing flavonoids is associated with a reduced risk of cardiovascular diseases (CVD), possibly by lipid-lowering effects. On the other hand, for one of these flavonoids, quercetin, also genotoxicity was shown especially in in vitro bioassays. Therefore, the first aim of this thesis was to identify mechanisms underlying potential beneficial health effects of flavonoids. The focus was on hepatic lipid metabolism and circulating lipids and a molecular and physiological approach was used. Secondly, we aimed to study the potential in vivo genotoxic effects of quercetin by transcriptome analyses in liver and small intestine, since these represent the tissues of first contact exposed to relatively high levels upon oral intake of flavonoids.
Circulating lipids are important CVD-related risk markers, which are in general determined with commercially available enzyme-based assays. However, the usual enzyme in these assays, peroxidase, has previously been reported to be inhibited by flavonoids. Therefore, we have studied in chapter 2 whether these assays can adequately be used in flavonoid research. We observed that various flavonoid aglycones interfere with peroxidase used in triglycerides (TG) and free fatty acids (FFA) enzymatic assays, reporting incorrect lower TG and FFA levels than actually present. Furthermore, addition of metabolites such as isorhamnetin or quercetin-3-O-glucuronide, the major metabolite of quercetin in human and rat plasma, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen towards reduced FFA levels. It can be concluded that when applying these biochemical assays, vigilance is needed and alternative analytical methods assessing FFA or TG levels should preferably be applied for studying the biological effects of flavonoids on TG and FFA levels.
In chapter 3 mechanistic and physiological effects of quercetin on hepatic lipid metabolism were studied. C57BL/6JOlaHsd male adult mice received a mild high-fat (30 en%) diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H-NMR were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify potential mechanisms underlying altered circulating lipid levels by quercetin supplementation. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, TG levels were decreased by 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) levels were increased by 13% (p<0.01). Levels of palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega-oxidation. Gene expression profiling showed indeed that quercetin increased hepatic lipid metabolism, especially omega-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450 activities) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up- regulated in the quercetin-fed mice. We concluded that quercetin intake increased hepatic lipid omega-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects of quercetin on CVD.
Subsequently, in chapter 4 effects of quercetin supplementation were studied in mice given a high-fat (40 en%) background diet. The set-up of the experiment was the same as in chapter 3, with the exception of the background diet that was used, which was different in fat content and composition. This high-fat diet-induced body weight gain, and serum and hepatic lipid accumulation, which are all known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the effects of the flavonoid quercetin on hepatic lipid metabolism in mice given this high-fat diet background. C57BL/6JOlaHsd male adult mice received the high-fat diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Body weight gain was 29% lower in quercetin fed mice versus control mice (p<0.01), while the energy intake was not significantly different. Quercetin supplementation lowered high-fat diet-induced hepatic lipid accumulation to 29% of the amount present in the control mice (p<0.01). 1H-NMR serum lipid profiling revealed that the supplementation also significantly lowered high-fat diet-induced increases in serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor Car, were down-regulated. However, the induction of omega-oxidation observed by quercetin supplementation to a mild high-fat (30en%) diet (chapter 3), was not observed this time with the high-fat (40en%) diet. Cumulatively, quercetin decreased high-fat diet-induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are considered to be under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content and composition of the diet.
In chapter 5 we investigated whether flavonoids from other flavonoid subclasses can exert the same effects as we observed for quercetin. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins, in C57BL/6JOlaHsd male adult mice fed a high-fat diet for 12 weeks were compared, relative to a normal-fat diet. High-fat diet-induced body weight gain was significantly lowered by all flavonoids (17-29%), but most by quercetin. Quercetin significantly lowered high-fat diet-induced hepatic lipid accumulation (by 71%). High-fat diet-induced increases of mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin, and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected.
The effects on body weight and adiposity could not be explained by individual significant differences in energy intake, energy expenditure, nor by differences in activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly down-regulated by all flavonoids. Overall, all five flavonoids lowered parameters of high-fat diet-induced adiposity, with quercetin being most effective.
Next to the beneficial health effects of flavonoids, the safety of flavonoids is under discussion, mainly because of potential genotoxic effects found for quercetin in vitro. Therefore, in chapter 6 the in vivo genotoxicity of this flavonoid was studied by transcriptome analyses in two tissues, small intestine and liver, where the highest exposure to quercetin is expected. This is especially of interest in view of high intake by widely available food supplements. Quercetin (0.33%) supplemented to a high-fat diet was administered to C57BL/6JOlaHsd male adult mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. General microarray pathway analysis of liver and small intestinal tissue samples showed no regulation of genotoxicity related pathways. In addition, analysis of DNA damage pathways in these tissues did also not point at genotoxicity. Furthermore, comparison with a published classifier set of transcripts for identifying genotoxic compounds did not reveal any similarities in the regulation of these classifier set by quercetin. Available microarray datasets of known genotoxic liver carcinogens, 2-acetylaminofluorene and aflatoxin B1 in mice were taken along as positive controls for comparison, and indeed showed genotoxic properties (regulation of genotoxic related genes) in the analyses. This transcriptomic analysis showed that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks did not induce genotoxicity in liver and small intestine.
In conclusion, we have shown in vivo efficacy of flavonoids reflected by effects on metabolic health parameters, including hepatic lipid metabolism. These effects on hepatic lipid metabolism seemed to be related or influenced by the transcription factor CAR. The dietary contexts appeared to modify the health effects. The five studied flavonoids in general showed the same effects, with quercetin being the most effective. No genotoxicity of quercetin was found by transcriptome analyses in liver and small intestine. Overall, we have obtained indications for beneficial health effects of flavonoids in mice, which makes it interesting to study if these effects can be extrapolated to humans to further explore their potential as functional compounds of dietary flavonoid intake.
Functional characterization of the PPAR targets ANGPTL4 and HILPDA in lipid metabolism
Mattijssen, F.B.J. - \ 2014
Wageningen University. Promotor(en): Sander Kersten. - Wageningen : Wageningen University - ISBN 9789461739087 - 193
lipidenmetabolisme - receptoren - genotype-voeding interactie - lipid metabolism - receptors - genotype nutrition interaction
The peroxisomal proliferator activator receptors (PPARs) are ligand-activated transcription factors that play important roles in the regulation of lipid metabolism. Three PPAR isoforms have been identified: PPARα, PPARβ/δ, and PPARγ. Each isoform has specific functions determined by their relative abundance in a cell as well as ligand specificity.
A highly sensitive PPAR target gene is represented by Angiopoietin-like 4 (ANGPTL4), which was discovered by two independent groups in 2000. ANGPTL4 is produced in a number of organs including liver and adipose tissue where its expression is governed by PPARα and PPARγ, respectively. Upon secretion, ANGPTL4 is cleaved into n- and c-terminal fragments that have divergent functions. nANGPTL4 is known to function as an inhibitor of lipoprotein lipase and hepatic lipase, whereas cANGPTL4 is involved in a number of processes including tumorigenesis and wound healing and is known to interact with integrins β1 and β5.
In this thesis we set out to expand our knowledge on the molecular function of ANGPTL4 in the regulation of lipid metabolism. We used a variety of animal models, cell culture, biochemical assays, and other functional measurements to zoom in on previously unexplored aspects of ANGPTL4.
Feeding mice deficient in ANGPLT4 a diet rich in long-chain saturated fatty acids elicited a complex phenotype and Angptl4-/- mice ultimately died from fibrinopurulent peritonitis. In contrast, the prevalence of the lethal phenotype was absent when the fat component of the high-fat diet was changed to medium-chain fatty acids, suggesting a role for increased chyle flow. Indeed, Angptl4-/- mice had dramatically enlarged mesenteric lymph nodes which contained numerous lipid laden macrophages. In vitro experiments showed that PPARβ/δ mediated induction of ANGPTL4 inhibits macrophage LPL. In the absence of ANGPTL4 there is increased lipid uptake in mesenteric lymph node macrophages, leading to ER stress and subsequent inflammatory response.
Additionally, Angptl4-/- mice gain more weight when fed a high-fat diet containing mainly unsaturated fatty acids. The increased body weight and adiposity was unrelated to food intake, activity, or energy expenditure. Remarkably, we observed increased lipid digestion in Angptl4-/- mice, which coincided with increased luminal lipase activity in the intestines of Angptl4-/- mice. Using biochemical assays we reveal that ANGPTL4 inhibits pancreatic lipase.
In the second part of this thesis we identified a novel PPAR target gene, hypoxia inducible lipid droplet associated (HILPDA). We observed HILPDA expression to be increased in liver slices exposed to a synthetic PPARα ligand. Additionally, oral dosing of similar ligand induced a marked increase in Hilpda expression in wild-type mice but not in Pparα-/- mice. PPAR mediated induction of Hilpda expression was found to be mediated by a conserved and functional PPRE located 1200 base pair or the transcription start site of HILPDA. Functional characterization of HILPDA in liver was performed via adeno-associated virus mediated overexpression. Interestingly, increased hepatic expression of HILPDA was associated with the development of a fatty liver, which could be attributes to a decrease in hepatic VLDL production.
HILPDA was also found to be highly expressed in both human and mouse adipose tissue, where its expression is under the control of PPARγ and β-adrenergic receptor. Moreover, adipose tissue HILPDA expression was increased with fasting and decreased with high-fat feeding. Despite the regulation of adipose tissue HILPDA by PPARγ, we observed no effect of HILPDA on adipogenesis. Furthermore, adipose tissue specific Hilpda knock-out mice showed no major metabolic perturbations upon fasting. However, overexpression of HILPDA in adipocytes significantly reduced the release of NEFA upon β-adrenergic receptor activation. Induction of HILPDA by β-adrenergic receptor stimulation may be part of feedback mechanism to regulate adipocyte lipolysis.
In conclusion, in thesis we have extended the current knowledge on the function of ANGPTL4. We show that ANGPTL4 serves as an important regulator in the process of lipid digestion and also in the protection of macrophages that reside in mesenteric lymph nodes that are exposed to high concentrations of lipid. HILPDA is a novel PPAR target that is involved in hepatic VLDL secretion and adipocyte lipolysis. Future research will focus on elucidating the mechanistic aspects of the regulation and function of HILPDA.
High fat challenges and detection of early perturbations in endothelial health : the use of a comprehensive phenotyping approach
Esser, D. - \ 2013
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Lydia Afman. - S.l. : s.n. - ISBN 9789461735911 - 168
endotheel - vetconsumptie - voedingsvet - lipidenmetabolisme - chocolade - endothelium - fat consumption - dietary fat - lipid metabolism - chocolate
Background:Cardiovascular disease (CVD) is one of the leading causes of morbidity and mortality worldwide. One of the pathophysiology’s that play a pivotal role in the development and progression of CVD is a dysfunctional endothelium. An important lifestyle risk factor for endothelial dysfunction is the diet and several nutrients have been classified to be either beneficial or harmful for the endothelium. Although CVD usually affects middle-aged or older adults, the onset of endothelial dysfunction begins in early life, emphasising the need for primary prevention. We therefore aimed to identify markers of early perturbations in endothelial health by using dietary stressors, e.g. high fat (HF) challenge test. Thereafter we aimed to evaluate if the potential early markers are reversible and can be improved after an intervention with a dietary anti-stressor.
Methods:First we validated the HF challenge test as a tool to trigger the endothelial response capacity. For that purpose, we compared the postprandial response after a HF shake with an average breakfast shake in young healthy men by assessing several plasma markers and functional measures of endothelial function. To identify new markers for early perturbations in endothelial health and to optimized the HF challenge test we applied three HF challenges differing in fatty acid type in two populations of middle-aged men, i.e. one at high- and one at low risk for developing CVD and characterized the postprandial response by applying high-throughput metabolomic and transcriptomic tools next to an extensive phenotyping of vascular function and vascular health parameters. Lastly, we evaluated if the, in the studies above, identified potential early biomarker profile is reversible and can be improved after an intervention with a dietary anti-stressor by means of a high flavanol chocolate intervention.
Results:In young men, we observed that a HF challenge decreased flow mediated dilation (FMD), but this decrease was also found after the consumption of an average breakfast shake. IL-8 concentrations were more pronouncedly increased after HF shake consumption compared to an average breakfast control shake. In middle-aged men, a HF challenge decreased the augmentation index (AIX) and elicited an activated state of cellular adherence in the circulation as determined by increased plasma soluble adhesion molecules, increased leukocyte cell surface integrin and selectin expression and increased number of leukocytes. A challenge high in mono-unsaturated fatty acids (MUFAs) elicited the highest postprandial triglyceride (TG) concentrations and the most pronounced effects on AIX. By applying high-throughput metabolomic tools, we observed that oxylipin profiles were affected by the HF challenge and that these changes were depended on dietary fatty acid composition. Application of transcriptome profiling revealed that changes in peripheral blood mononuclear cell (PBMC) gene expression profiles after a HF challenge test were different between lean and obese subjects, with the most deviating effect after MUFA intake. The saturated fatty acid (SFA) shake decreased the expression of genes involved in cholesterol uptake and cholesterol biosynthesis and increased expression of genes involved in cholesterol efflux. MUFA increased expression of inflammatory genes and of peroxisome proliferator-activated receptor α (PPARα) target genes involved in β-oxidation. 4-week daily intake of a dietary anti-stressor, e.g. dark chocolate, increased fasting FMD and decreased AIX, and elicited a less activated state of cellular adherence, as determined by a decrease in plasma soluble adhesion molecules, a decrease in leukocyte cell surface integrin and selectin expression and a decrease in the number of leukocytes.
Conclusions:In this thesis we extensively characterized the postprandial response to a HF challenge in human subjects with different disease risk profiles and optimized the HF challenge test. We identified MUFAs as most potent fatty acids to trigger the vascular and cellular response capacity, which makes it the optimal fatty acid type to use in a HF challenge test. We demonstrated that besides functional measures of vascular function, also plasma and cellular factors involved in leukocyte adhesion to the endothelium are adversely affected by dietary stressors and are beneficially affected by a dietary anti-stressor. Therefore, we conclude that endothelial health can be more comprehensively measured by means of a biomarker profile consisting not only of the vascular function measures FMD and AIX, but also of a subset of soluble adhesion molecules in the plasma, leukocyte counts and cell surface integrin and selectin expression. To identify potential new leads for biomarkers, we applied whole genome gene expression profiling, combined with the HF challenge test which enabled us to detect small differences in health status. Furthermore, we identified metabolic and inflammatory pathways that are specifically affected by either MUFAs or SFAs. These findings increased our understanding on how a SFA or MUFA challenge exert their distinct effects on stress related and metabolic compensatory cellular processes and provided us with new potential leads to detect early perturbations in endothelial health.
Nutritional Systems Biology of Fat : integration and modeling of transcriptomics datasets related to lipid homeostasis
Ohid Ullah, M. - \ 2012
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Guido Hooiveld. - S.l. : s.n. - ISBN 9789461733818 - 158
vetzuren - genexpressie - lipidenmetabolisme - obesitas - transcriptomica - statistische analyse - wiskundige modellen - fatty acids - gene expression - lipid metabolism - obesity - transcriptomics - statistical analysis - mathematical models
Fatty acids, in the form of triglycerides, are the main constituent of the class of dietary lipids. They not only serve as a source of energy but can also act as potent regulators of gene transcription. It is well accepted that an energy rich diet characterized by high intakes of dietary fat is linked to the dramatic increase in the prevalence of obesity in both developed and developing countries in the last several decades. Obese individuals are at increased risk of developing the metabolic syndrome, a cluster of metabolic abnormalities that ultimately increase the risk of developing vascular diseases and type 2 diabetes. Many studies have been performed to uncover the role of fatty acids on gene expression in different organs, but integrative studies in different organs over time driven by high throughput data are lacking. Therefore, we first aimed to develop integrative approaches on the level of individual genes but also pathways using genome-wide transcriptomics datasets of mouse liver and small intestine that are related to fatty acid sensing transcription factor peroxisome proliferator activated receptor alpha (PPARα). We also aimed to uncover the behavior of PPARαtarget genes and their corresponding biological functions in a short time series experiment, and integrated and modeled the influence of different levels of dietary fat and the time dependency on transcriptomics datasets obtained from several organs by developing system level approaches.
We developed an integrative statistical approach that properly adjusted for multiple testing while integrating data from two experiments, and was driven by biological inference. By quantifying pathway activities in different mouse tissues over time and subsequent integration by partial least squares path model, we found that the induced pathways at early time points are the main drivers for the induced pathways at late time points. In addition, using a time course microarray study of rat hepatocytes, we found that most of the PPARα target genes at early stage are involved in lipid metabolism-related processes and their expression level could be modeled using a quadratic regression function. In this study, we also found that the transcription factorsNR2F, CREB, EREF and RXR might work together with PPARα in the regulation of genes involved in lipid metabolism. By integrating time and dose dependent gene expression data of mouse liver and white adipose tissue (WAT), we found a set of time-dose dependent genes in liver and WAT including potential signaling proteinssecreted from WAT that may induce metabolic changes in liver, thereby contributing to the pathogenesis of obesity.
Taken together, in this thesis integrative statistical approaches are presented that were applied to a variety of datasets related to metabolism of fatty acids. Results that were obtained provide a better understanding of the function of the fatty acid-sensor PPARa, and identified a set of secreted proteins that may be important for organ cross talk during the development of diet induced obesity.
Genetics and genomics of cholesterol and polyunsaturated fatty acid metabolism in relation to coronary heart disease risk
Lu Yingchang (Kevin), Y. - \ 2011
Wageningen University. Promotor(en): Edith Feskens; Michael Muller, co-promotor(en): J.M.A. Boer. - [S.l.] : S.n. - ISBN 9789461730350 - 216
lipidenmetabolisme - cholesterol - meervoudig onverzadigde vetzuren - hartziekten - risicoschatting - genetische factoren - nutrigenomica - lipid metabolism - cholesterol - polyenoic fatty acids - heart diseases - risk assessment - genetic factors - nutrigenomics
Coronary heart disease (CHD) continues to be a leading cause of morbidity and mortality among adults worldwide. Deregulated lipid metabolism (dyslipidemia) that manifests as hypercholesterolemia, hypertriglyceridemia, low high-density-lipoprotein (HDL) cholesterol levels or a combination of those, is an established risk factor for CHD among other established risk factors. Linoleic acid (LA, C18:2n-6) and alpha-linolenic acid (ALA, C18:3n-3) are polyunsaturated fatty acids (PUFAs) that cannot be synthesized de novo by human or animal cells, and therefore must be obtained from the diet. From these two PUFAs, two series of long-chain PUFAs are formed; the omega-6 series that are synthesized from LA, and the omega-3 series that are from ALA. Formation of these long-chain PUFAs involves a series of alternate desaturation and elongation processes. These PUFAs, especially, omega-3 PUFAs, have long been observed to reduce CHD risk. In contrast to the consistently observed cardiovascular protective effects of omega-3 PUFAs, accumulating evidence suggests a potential pro-atherogenic effects of omega-6 PUFAs, which is now still under debate.
It has been estimated that genetic factors account for 26%-69% of inter-individual variation in CHD risk. These genetic factors are thought to influence CHD risk both directly and through effects on known CHD risk factors such as plasma lipid levels. The heritability of plasma lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides (TG)) is estimated to be about 50% (ranging from 28%-78%). With the success of recent genome-wide association studies (GWAS), many genetic variants underlying intermediate risk factors of CHD (including plasma lipid levels) and CHD itself have been identified. Whether this new genetic information could be used to improve CHD risk prediction is still marginally explored, and for some variants, the underlying mechanisms for their mediated effects on CHD risk are still unknown. The aim of this research is to investigate common genetic determinants of plasma lipid levels (cholesterol and polyunsaturated fatty acid levels) using a pathway-driven approach, and to explore whether such common genetic variants could be used to improve CHD prediction using a population based genetic approach. An additional aim was to explore the underlying mechanisms of cardiovascular protective effects of PUFAs using a genomic approach.
In order to explore whether common genetic variants are involved in determining plasma cholesterol levels, we used data from 3575 men and women from the Doetinchem cohort, which was examined thrice over 11 years. They were genotyped on 384 single nucleotide polymorphisms (SNPs) across 251 genes in regulatory pathways that control fatty acid, glucose, cholesterol and bile salt homeostasis.
In order to explore whether common genetic variants could be used to predict future CHD risk,we used the data from CAREMA cohort that involved 15,236 middle-aged subjects and was followed up for a median of 12.1 years. 179 SNPs associated with CHD or its risk factors in GWAS published up to May 2, 2011 were genotyped in the 2221 subcohort members and 742 incident CHD cases. In addition, fatty acids from plasma cholesteryl esters were quantified in 1323 subcohort members and 537 CHD cases. They were used to explore whether δ-5 and δ-6 desaturase activities were associated with CHD risk.
In order to perform a comparative analysis of the effects of fenofibrate and fish oil at transcriptome and metabolome level, 34 mice were randomized by weight-matching into three groups (n = 10 in control group, and n = 12 in fenofibrate or fish oil intervention group), and fed a research diet supplemented with sunflower oil (containing 81.3% oleic acid, 7% energy intake) in control group, sunflower oil (7% energy intake) and fenofibrate (0.03% w/w) in fenofibrate group, and fish oil (Marinol C-38 fish oil: 23.1% EPA and 21.1% DHA, 7% energy intake) in fish oil group for 2 weeks. At the end of treatment, mice were fasted with drinking water available, and were subsequently sacrificed by cervical dislocation under isoflurane anesthesia. Blood was collected via orbital puncture. Livers were dissected, directly frozen in liquid nitrogen and stored at −80°C until further analysis. Microarray analysis was performed on individual mouse livers. The LC-MS method was used for measuring plasma lipids and non-esterified free fatty acids, and the GC-MS method was used for measuring a broad range of metabolites.
In chapter 2, 3, and 4, common genetic variants in the genes along known cholesterol metabolic pathways, such as bile acid and bile metabolic pathways, the HDL cholesterol metabolic pathway, and the plasma total cholesterol metabolic pathway, are involved in determining plasma cholesterol levels. The modest effect associated with each individual variant, however, caused the amount of heritability explained by them in aggregate to be relatively small: 13 single nucleotide polymorphisms (SNPs) explained 4% of inter-individual variation in HDL cholesterol levels (Chapter 3), whereas 12 SNPs explained 6.9% of inter-individual variation in total cholesterol levels (Chapter 4).
In chapter 5, we found that genetic variants in the FADS1 gene potentially interact with dietary PUFA intake to affect plasma cholesterol levels. A high intake of omega-3 PUFAs was associated with increased plasma non-HDL cholesterol levels, consistent with increased plasma LDL cholesterol levels observed in fish oil intervention studies. Increased LDL cholesterol levels could be due to hepatic downregulation of the LDL receptor gene (LDLR) in subjects with high omega-3 PUFA intakes. This is further confirmed by the findings described in Chapter 6 that the hepatic LDLR gene was significantly downregulated in fish oil treated mice. This study also confirmed PUFAs to be weak PPAR ligands. The increased plasma HDL cholesterol levels in the subjects with high PUFA intakes in Chapter 5 could be due to PPARs-mediated genes that are directly involved in HDL lipoprotein metabolism. All these may explain the changes in blood cholesterol levels upon PUFA intake observed in human studies.
In Chapter 6, we found that not only downregulation in the hepatic lipogenic pathway but also upregulation in hepatic fatty acid oxidation pathways are involved in lowering plasma TG levels upon fish oil treatment. The striking parallel between fenofibrate and fish oil in hepatic downregulation of blood coagulation and fibrinolysis pathways suggest that hepatic activation of PPARα is potentially one of the mechanisms responsible for anticoagulation effects of fish oil treatment observed in humans.
In Chapter 7, with confirmed effects of rs174547 in FADS1 on PUFA levels and δ-5 desaturase activities and also protective effects of DHA on CHD, we observed a reduced CHD risk of increased δ-5 desaturase activity. Increased δ-5 desaturase activity could contribute to the intracellular increase of EPA and especially arachidonic acid (C20:4n-6) levels. Despite the potential pro-coagulant and pro-inflammatory effects of increased exposures of arachidonic acid and its derived eicosanoid metabolites, there is no evidence of increased CHD risk with increased habitual arachidonic acid intake so far. Some of the oxygenated metabolites of arachidonic acid were found to have anti-inflammatory and pro-resolving actions. High dietary n-6 PUFA intakes or high plasma n-6 PUFA levels are associated with increased blood HDL cholesterol levels and reduced TG (or VLDL particle) levels. All these point to a potential cardiovascular protective effect of n-6 PUFAs. The fact that increased EPA and/or DHA levels associated with increased δ-5 desaturase activity protect against CHD is consistent with the current established cardiovascular protective effect of increased n-3 PUFA exposure, especially EPA and DHA.
In Chapter 8, the current known common genetic variants associated with CHD risk factors (blood pressure, obesity, blood lipid levels, and type 2 diabetes) and CHD itself from published GWAS are examined to see whether they provide additional value in CHD risk prediction beyond established traditional CHD risk factors. We constructed several gene risk scores (GRS) for CHD that consisted of SNPs directly associated with CHD or intermediate CHD risk factors in GWAS, and tested their relationship to incident CHD and their potential to improve risk prediction. The weighted GRS based on 29 CHD SNPs predicted future CHD independently from established traditional risk factors. However, the GRS based on 153 SNPs associated with intermediate risk factors and the GRS based on the total 179 SNPs did not. None of them improved risk discrimination. Risk classification of CHD, measured by the net reclassification index, improved only when the GRS based on the 29 CHD SNPs was used. These results are generally consistent with the results from other recent studies that took a similar approach as ours. However, the final conclusions on GRS application could not be drawn at this early stage. With a great understanding of the genetic architecture of CHD in the future, more research should be done on this topic.
Our studies in this thesis demonstrated that common genetic variants along the known candidate cholesterol metabolic pathways are involved in determining the plasma cholesterol levels. PUFAs are not only weak PPARα ligands, but also inhibit SREBPs’ activities. All these could explain part of the cardiovascular protective effects (increased HDL cholesterol levels and reduced TG levels) of PUFAs, increased LDL cholesterol levels upon fish oil treatment in humans, and potentially reduced CHD risk of high δ-5 desaturase activities. At present, many questions remain about the feasibility of genetic risk prediction of CHD. Clinicians should continue to inquire about family history of CHD for risk prediction, because this represents a simple, cheap, and useful risk factor for CHD that likely represents the net integrated effects from hundreds of genetic risk variants.
PPARa: master regulator of lipid metabolism im mouse and human : identification of hepatic PPARa target genes by expression profiling
Rakhshandehroo, M. - \ 2010
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Sander Kersten. - [S.l. : S.n. - ISBN 9789085857716 - 238
lipidenmetabolisme - transcriptiefactoren - genexpressie - muizen - mens - nutrigenomica - lipid metabolism - transcription factors - gene expression - mice - man - nutrigenomics
The peroxisome proliferator activated receptor alpha (PPARα) is a ligand activated tran- scription factor involved in the regulation of a variety of processes, ranging from inflam- mation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid derived compounds. PPARα governs biological processes by altering the expression of large number of target genes. Although the role of PPARα as a gene regulator in liver has been well estab- lished, a comprehensive overview of its target genes has been missing so far. Additionally, it is not very clear whether PPARα has a similar role in mice and humans and to what extent target genes are shared between the two species. The aim of the research presented in this thesis was to identify PPARα-regulated genes in mouse and human liver and thereby further elucidate hepatic PPARα function. The applied nutrigenomics approaches are mainly expression microarrays combined with knockout mouse models and in vitro cell culture systems. By combining several independent nutrigenomics studies, we generated a comprehensive overview of PPARα-regulated genes in liver with the focus on lipid metabolism. We identi- fied a large number of PPARα target genes involved in different aspects of lipid metabolism. Furthermore, a major role of PPARα in lipogenesis was detected. Our data pointed to several novel putative PPARα target genes. Next, we compared PPARα-regulated genes in primary mouse and human hepatocytes treated with the PPARα agonist Wy14643 and generated an overview of overlapping and species specific PPARα target genes. A large number of genes were found to be regulated by PPARα activation in human primary hepatocytes, which iden- tified a major role for PPARα in human liver. Interestingly, we could characterize mannose binding lectin 2 (Mbl2) as a novel human specific PPARα target gene. Plasma Mbl2 levels were found to be changed in subjects receiving fenofibrate treatment or upon fasting. Regula- tion of Mbl2 by PPARα suggests that it may play a role in regulation of energy metabolism, although additional research is needed. We also compared the PPARα-induced transcriptome in HepG2 cells versus primary human hepatocytes to investigate the suitability of HepG2 cells in PPARα research. The results re- vealed that the HepG2 cell line poorly reflects the established PPARα target genes and func- tion, specifically with respect to lipid metabolism. Finally, we characterized the transcription factors Klf10 and Klf11 as novel PPARα target genes. Our preliminary findings using in vitro transfection assays and in vivo tail vein injection of plasmid DNA suggested a potential metabolic role of Klf10 and Klf11 in liver. In conclusion, this thesis has extended our understanding of PPARα-regulated genes and function in liver, and has specifically highlightened a major role of PPARα in human hepa- tocytes. This research has also given birth to a possible biomarker of hepatic PPARα activity which is of great interest for future studies. Considering the need for proper biomarkers in the field of nutrigenomics and beyond, the properties of Mbl2 as a biomarker should be further investigated. The identification of other novel putative PPARα target genes offers ample op- portunities for continued research.
Genomic, transcriptomic and proteomic analysis of the nuclear receptor PPARa
Meer, D.L.M. van der - \ 2010
Wageningen University. Promotor(en): Sacco de Vries; Michael Muller, co-promotor(en): Sander Kersten. - [S.l. : S.n. - ISBN 9789085855552 - 128
biochemische receptoren - lipidenmetabolisme - transcriptiefactoren - genexpressieanalyse - transcriptomics - eiwitexpressieanalyse - biochemical receptors - lipid metabolism - transcription factors - genomics - transcriptomics - proteomics
Accumulating wealth in the western world has led to an increase in chronic health problems such as obesity. Obesity is often associated with abnormal metabolic function, resulting in increased glucose, cholesterol and triglyceride levels in the blood. Development of cardiovascular diseases and type 2 diabetes is often observed as a result of obesity. Disturbed lipid metabolism is one of the major processes altered with obesity. Investigation of the fundamental molecular mechanisms involved in lipid metabolism may be beneficial to the prevention and treatment of this disease. One of the proteins involved in the regulation of the lipid metabolism is the nuclear receptor named Peroxisome Proliferator-Activated Receptor alpha (PPARα). PPARα resides in the nucleus and functions as an inducible transcription factor, which upon binding of fatty acid binds to specific regions within the DNA. As a consequence, the transcription of several genes that encode for enzymes and other proteins that are responsible for the catabolism of fatty acids and energy homeostasis is increased.
In this thesis, several aspects related to the mechanism of PPARα function are described. First it was investigated what proteins physically interact with PPARα and may thus be necessary for proper transcription of target genes. Secondly it was investigated what genomic locations PPARα is bound to and if the binding results in enhanced expression of genes located near these binding locations. Thirdly, the predictability of PPARα DNA binding based on specific DNA sequences was studied. Finally, a comparison was made between PPARα regulated genes in primary human hepatocytes and the human liver cell line HepG2.
The presence of PPARα alone is not sufficient to induce the expression of a target gene. Additional proteins, coactivators, have to interact with PPARα to form a larger protein complex that stimulates gene expression. Currently, it is not completely known which coregulator proteins are involved in PPARα-mediated gene regulation. With the use of immunoprecipitation and mass spectrometry, it is possible to isolate protein complexes and identify protein interactions with a tagged protein. Those techniques were used to identify proteins that interact with PPARα, resulting in the identification of several proteins that may play a role in the regulation of gene expression by PPARα.
To investigate which genomic locations are bound by PPARα after ligand activation, the ChIP-Chip technology was used. This technique combines chromatin immunprecipitation with specialized microarrays called DNA tiling arrays . With the use of ChIP it is possible to isolate DNA fragments of approximately 1000 base pairs that are bound by PPARα. By hybridizing the isolated DNA fragments to a promoter tiling microarray, it is possible to create a profile of PPARα binding across the whole genome. Over 4000 DNA regions were identified using this approach, including binding regions near genes that were previously described to be regulated by PPARα The identified genes located near PPARα binding regions were compared with gene expression data. PPARα binding regions near genes that were transcriptionally downregulated were found to be enriched for a binding motif for the STAT transcription factors. Binding of STAT3 and STAT1 to these regions was shown to be reduced upon PPARα activation. Interestingly, STATs are involved in inflammatory processes that are known to be inhibited by PPARα activation and were previously shown to be inhibited by PPARγ. Accordingly, it can be hypothesized that down-regulation of gene expression by PPARα ligands is partially mediated by interfering with binding of STAT3 and STAT1 to the DNA. Similarly, PPARα binding regions near genes that were transcriptionally upregulated were found to be enriched for a binding motif for TBP and C/EBPα. In addition, important cross-talk between PPARα and SREBP was found with respect to upregulation of gene expression. Possibly, these combinations of transcription factors play an important role in PPARα-mediated gene regulation.
To examine the relation between DNA binding by PPARα, changes in gene expression, and predicted PPARα DNA binding, we compared various data sets via a systems biology type of analysis. This comparison clearly showed that the number of locations where PPARα is able to bind greatly exceeds the number of genes that are transcriptionally regulated. This indicates that in many cases DNA binding by PPARα does not result in changes in expression of genes located nearest to the binding location. Possibly, PPARα binding has another unknown function at those locations. It appeared also from this comparison that a prediction of PPARα binding on the DNA on the basis of sequence alone did not give an enrichment of PPARα binding sites in the identified ChIP-Chip regions compared to control promotor regions. However, one of the prediction algorithms did give an enrichment on basis of sequence when only considering PPARα binding locations associated with upregulated genes. The latter method may be useful for identification of new genes that are regulated by PPARα.
Finally, a comparison was made between the gene expression patterns after PPARα activation in the human liver cell line HepG2 and human primary hepatocytes. It appears that the HepG2 cell line poorly reflects the known PPARα function in comparison to primary hepatocytes. Several known PPARα target genes are induced by PPARα agonist in primary hepatocytes but are unresponsive in the HepG2 cell line. Therefore, it is recommended to exercise caution in the interpretation of results using HepG2 cells and if possible select primary hepatocytes. However, since an alternative human liver cell line is not widely available, HepG2 still remains the best model for several applications.
Functional characterization of Angptl4 protein
Lichtenstein, L.L. - \ 2009
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Sander Kersten. - [S.l. : S.n. - ISBN 9789085855033 - 118
vetzuren - genexpressie - lipidenmetabolisme - vasten - nutrigenomica - fatty acids - gene expression - lipid metabolism - fasting - nutrigenomics
Background: Elevated plasma triglycerides (TG) are increasingly recognized as a risk factor for atherosclerosis. A new adipocytokine was discovered by several groups which is referred to as Angptl4 (Angiopoietin-like protein 4). Angptl4 was recently identified as a major determinant of plasma TG levels in mice. Angptl4 is a 50 KDa secreted protein belonging to the family of fibrinogen/angiopoietin-like proteins.
Objective/Design: In order to characterize the metabolic function of Angptl4, we explored dietary modulation effects on transgenic mice overexpressing Angptl4.
Results: Taking advantage of the induction of Angptl4 by fasting, we showed that Angptl4 strongly inhibits LPL and HL activities. Later, we reported the development, validation and utilization of an ELISA assay to quantitatively assess Angptl4 levels in human plasma. Within an individual, Angptl4 levels rise in response to elevation of plasma free fatty acids. Furthermore, we address the function of Angptl4 in the heart. Dietary unsaturated fatty acids have a major impact on human health, which is likely achieved via changes in gene expression. Fatty acids regulate gene expression mainly via nuclear receptors, including the PPARs. Angptl4 appears to be regulated by dietary fat in a PPARb/d dependent, but in a PPARa independent way. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma TG-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. Finally we investigated the function of Angptl4 in the intestine. Elevated saturated fat consumption is associated with increased risk for numerous chronic diseases, including inflammatory bowel disease, and obesity. Besides carrying out nutrient digestion and absorption, the GI-tract also produces a variety of hormones that play pivotal roles in nutrient handling and energy homeostasis. We report that Angptl4 is fat sensitive hormone produced by enteroendocrines cells. Noticeably, chronic saturated fat feeding led to complex and dramatic phenotype in Angptl4-/- mice. Angptl4-/- fed saturated fat developed a severe pathology leading to death. This lethal phenotype is preceded by excessive inflammation as shown by a dramatic hepatic acute phase response.
Conclusion: The data show that Angptl4 protects against the pro-inflammatory and ultimately lethal effects of chronic overconsumption of saturated fat.
Exploring the activation and function of PPARa and PPARß/d using genomics
Sanderson-Kjellberg, L.M. - \ 2009
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Sander Kersten. - [S.l. : S.n. - ISBN 9789085854555 - 200
lipidenmetabolisme - lever - vetzuren - genexpressie - nutrigenomica - lipid metabolism - liver - fatty acids - gene expression - nutrigenomics
For many tissues fatty acids represent the major source of fuel. In the past few decades it has become evident that in addition to their role as energy substrates, fatty acids also have an important signaling function by modulating transcription of genes. An important group of transcription factors involved in mediating the effects of dietary fatty acids on gene transcription are the Peroxisome Proliferator-Activated Receptors (PPARs). PPARs are members of the superfamily of nuclear hormone receptors and regulate genes involved in numerous important biological processes, ranging from lipid metabolism to inflammation and wound healing. In the liver the dominant PPAR isoform has been show to be PPARα, although PPARβ/δ and PPARγ are expressed in liver as well.
The aim of this thesis was to further characterize the role of PPARα and PPARβ/δ in hepatic metabolism and study their activation by fatty acids. Even though PPARα as gene regulator in liver has been well described, a complete overview of its target genes has been lacking so far. By combining several nutrigenomics tools, we succeeded in creating a comprehensive list of PPARα-regulated genes involved in lipid metabolism in liver. Additionally, by using a unique design where mice were fed synthetic triglycerides consisting of one type of fatty acid, we could distinguish between different types of dietary unsaturated fatty acids in their ability to activate PPARα. Although it is well known that PPARα plays an important role in liver during fasting, no direct in vivo evidence exists that circulating free fatty acids are able to ligand activate hepatic PPARα. In our studies, we found that upregulation of gene expression by PPARβ/δ is sensitive to circulating plasma free fatty acids whereas this is not the case for PPARα. Not much is known about the function of PPARβ/δ in the liver. In order to better understand the role of this nuclear receptor, we compared the effects of PPARα and PPARβ/δ deletion on whole genome gene regulation and plasma and liver metabolites. Our results revealed that PPARβ/δ does not mediate an adaptive response to fasting, and pointed to a role for PPARβ/δ in hepatic glucose- and lipoprotein metabolism.
In conclusion, this thesis contributes to the important work of mapping the molecular mechanisms dictating lipid metabolism in the liver. By using several nutrigenomics tools, we are able to show that PPARα is a key mediator of the effect of dietary fatty acids on hepatic gene expression. In addition, we better define the roles of PPARα and PPARβ/δ in hepatic metabolism and provide a new concept for functional differentiation between PPARs in liver.
Fecundity in relation to lipid content in North Sea herring
Schouten, M. - \ 2007
IJmuiden : IMARES (Report / IMARES 07.012) - 37 p.
haringen - vruchtbaarheid - eierproductie - lipidenmetabolisme - lipiden - periode van kuitschieten - vissen - noordzee - herrings - fertility - egg production - lipid metabolism - lipids - spawning season - fishes - north sea
Characterization of the two PPAR target genes FIAF (Fasting-Induced Adipose Factor) and G0S2 (G0/G1 switch gene 2)
Zandbergen, F.J. - \ 2006
Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Sander Kersten. - s.l. : S.n. - ISBN 9789085043928 - 136
lipidenmetabolisme - obesitas - vetweefsel - genexpressie - vasten - hormonen - lipid metabolism - obesity - adipose tissue - gene expression - fasting - hormones
The prevalence of obesity has increased dramatically over the last decades. Obesity, defined as excess body fat, develops if energy expenditure is lower than its intake and if the surplus energy is stored in adipose tissue as fat. Excess adipose tissue, especially around the waist, is associated with an increased risk for diseases such as type 2 diabetes and atherosclerosis.These disorders aremajor causes of death from cardiovascular disease in the Western world.
Common features of obesity, atherosclerosis and diabetes are insulin resistance and elevated plasma levels of triglycerides (TG) and low-density lipoprotein (LDL) cholesterol, whereas high-density lipoprotein (HDL) cholesterol is decreased.
A number of the pharmacological interventions to treat early stages of atherosclerosis and type 2 diabetes target the peroxisome proliferator-activated receptors (PPARs). Activation of these transcription factors results in the expression of a variety of target genes, many of which play important roles in lipid metabolism. There are three PPAR isoforms: PPARa, PPARbor PPARd,and PPARg. Synthetic ligands for PPARaand forPPARgdecrease plasma TG levels and lower the concentration of LDL-cholesterol in blood whereas they elevate plasma HDL-cholesterol levels. Linked to their hypolipidaemic effect, they may also have hypoglycaemic effects, reducing chronically elevated insulin signalling and associated insulin resistance, which predisposes to the development of type 2 diabetes.
In an effort to gain more insight into the relationship between PPAR target gene expression and its beneficial effect on lipid metabolism with regard to atherosclerosis and type 2 diabetes, the expression of genes in liver of wild-type mice and mice that lack functional PPARα was compared during fasting. Amongthe genes that were found to be differentially regulated in the wild-type and the PPARα mice, wereboththefasting-induced adipose factor (FIAF) and the G 0 /G 1 switch gene 2 (G0S2) strongly up-regulated in the wild-type mice during fasting.
The research described in this thesis focuses on the characterization and elucidation of the function of these two genes and their protein products. FIAF belongs tothe family of fibrinogen/angiopoietin-like proteins and was previously found to be highly expressed in adipose tissue and to be up-regulated in response to fasting, hence its name. For G0S2, which was also highly expressed in adipose tissue and which we found to be localized to the endoplasmic reticulum (ER), no homologous genes could be found. During adipogenesis, the differentiation of pre-adipocytes into fully differentiated adipocytes, the levels of mRNA and protein for FIAF and G0S2 were greatly up-regulated.Subsequent experiments indicated that G0S2 is a direct PPARγ and probable PPARα target gene with a functional PPRE (PPAR-responsive element) in its promoter. Using the same approach, a functional PPRE was found within intron 3 of the FIAF gene, establishing FIAF as being a direct PPAR target gene too.
The up-regulation of G0S2 mRNA during the differentiation of adipocytes seemed to be specific for adipogenesis, no up-regulation of G0S2 mRNA was observed during osteogenesis or myogenesis.Furthermore, G0S2 expression was associated with cell cycle arrest in3T3-L1pre-adipocytes, which is required for the differentiation of these cells into adipocytes.This indicates that G0S2 may be involved in adipocyte differentiation.
Further investigation showed thatFIAF was present as the native protein and in truncated forms in bothmouse and human blood plasma. Interestingly,truncated FIAF was produced by human liver and treatment with PPARα agonist markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. The levels of both truncated and native FIAF showed marked inter-individual variation but were not associated with body mass index and were not influenced by prolonged semistarvation.
To determine the physiological role of FIAF, we studied the effect of FIAF overexpression in a transgenic mouse model (FIAF-tg mice). Thetransgenic mice had markedly reduced adipose tissue stores compared to their wild-type littermates, despite similar food intake. The FIAF-tg mice also had elevated plasma levels of TG, glycerol, free fatty acids (FFA), and HDL as well as very low-density (VLDL) cholesterol. The increase of plasma TG levels was attributable to elevated VLDL levels. Oral lipid loading showed that the FIAF-tg mice had severely impaired plasma TG clearance. The effects on plasma TG levels are most likely the result of FIAF-mediated inhibition of the activity of lipoprotein lipase (LPL), a key regulator of plasma TG clearance. The elevated levels of FFA and glycerol are indicative of increased lipolysis, a notion supported by theincreased expression level of adipose triglyceride lipase (ATGL) in the adipose tissue of FIAF-tg mice. Additional genes that were differentially expressed are involved in oxidative metabolism and uncoupling, which might explain the decreased weight of the FIAF-tg mice while their food intake was similar to that of their wild-type littermates.The elevated HDL levels might be the result of FIAF-mediated inhibition of other lipases in addition to LPL, e.g.endothelial and hepatic lipase (EL/HL).
Interestingly, after fractionation of mouse plasma by FPLC, the full length form of FIAF was present specifically in the HDL-containing fractions, whereas the truncated form of FIAF was specifically present in the LDL-containing fractions.In human plasma, both full length and truncated FIAF were only present in the HDL-containing fractions. In addition, the levels of truncated FIAF and HDL-cholesterol in human plasma correlated positively. Combined with our earlier finding that treatment with synthetic PPARaligandincreased the plasma levels of truncated FIAF in humans, this raises the possibility that FIAF might be involved in the mechanism by which PPARaligand treatment increases HDL-cholesterol levels in humans, resulting in a protective effect on atherosclerosis.
The up-regulation of FIAF during fasting and the ability to inhibit plasma TG clearance indicate that FIAF might play an important role in repartitioning TG from adipose tissue to other tissues under circumstances of energy shortage. In addition, alterations in FIAF signalling might be involved in dyslipidemia, the presence of abnormal lipid levels in the blood. FIAF thus forms an interesting candidate for therapeutic targeting of dyslipidemia.
Variation in sensitivity to fungicides which inhibit ergosterol biosynthesis in wheat powdery mildew.
Waard, M.A. de; Kipp, E.M.C. ; Horn, N.M. ; Nistelrooy, J.G.M. van - \ 1986
Netherlands Journal of Plant Pathology 92 (1986). - ISSN 0028-2944 - p. 21 - 32.
triticum aestivum - tarwe - hexaploïdie - gewasbescherming - fungiciden - pesticiden - nadelige gevolgen - selectiviteit - toxiciteit - onbedoelde effecten - niet-doelorganismen - plantenziekteverwekkende schimmels - lipidenmetabolisme - erysiphales - meeldauw - triticum aestivum - wheat - hexaploidy - plant protection - fungicides - pesticides - adverse effects - selectivity - toxicity - nontarget effects - nontarget organisms - plant pathogenic fungi - lipid metabolism - erysiphales - mildews
Gegevens van onderzoek naar de variatie in gevoeligheid voor fungiciden die de ergosterolbiosynthese remmen bij tarwemeeldauw. Sinds 1978 worden deze ungiciden in Nederland gebruikt bij de bestrijding van tarwemeeldauw