Peroxisomal and mitochondrial enzymes involved in lipid metabolism : studies on function and regulation

Fatty acids constitute a major part of the energy that we obtain from the diet and are also the principal source for mammals to store energy. To use the incoming or stored fatty acids as energy, the fatty acids needs to be metabolized of which the majority of fatty acids will be degraded by the mitochondrial β-oxidation system that in the end generates energy to the cell in the form of ATP. However, this organelle is not able to handle all kinds of fatty acids of which very long chain fatty acids, long chain methylbranched fatty acids and dicarboxylic acids are such cumbersome fatty acids. Therefore a second organelle, the peroxisome, is required for metabolism of these particular fatty acids. Also peroxisomes contain a β-oxidation system and similar to the mitochondrial system is the initial substrate a CoA-esterified fatty acid, so-called acyl-CoA. This thesis will focus on some enzymes that are active on these acyl-CoA esters, but that are not directly involved in the β-oxidation per se. Instead they contribute to the regulation of both acyl-CoA and free coenzyme A levels in different cellular compartments. This thesis will also include how these fatty acid degrading systems can be regulated at gene level by affecting different transcription factors by dietary ligands and by fasting. The peroxisomal Nudix hydrolase 7α (NUD7α), previously believed to be a CoASH degrading enzyme, was demonstrated to be a medium chain diphosphatase, most active on medium chain acyl-CoA esters, to produce 3’,5’-ADP and the corresponding 4’- acylphosphopantetheine thereof. NUDT7α expression and activity was down regulated by PPARα activation, which would prevent CoASH degradation and support a high rate of the β-oxidation in peroxisomes during these conditions. Peroxisomes are not only needed for the degradation of complex lipids, but are also essential for many other metabolic pathways such as bile acid and etherphospholipid synthesis and the degradation of D-amino acids and glyoxylate. The expression of gene transcripts that code for the proteins involved in these peroxisomal pathways was investigated almost throughout the whole mouse body with the aim to map the tissue expression of these pathways. The peroxisomal β-oxidation system is present in all examined tissues, however with differences in magnitude. More specifically expressed pathways are e.g. glyoxylate and D-amino acid degradation pathways. Auxiliary enzymes to the peroxisomal β-oxidation showed tissue specific expression, suggesting a high degree of tissue specific metabolite patterns, also being dependent on the metabolic state. The study also shows that PPARα is of major importance for the regulation in liver of the peroxisomal “transcriptome” during fasting. Mitochondria degrade both fatty acids and amino acids and the mitochondrial acyl- CoA thioesterase 9 (ACOT9) was shown to hydrolyze both long chain acyl-CoAs as well as short chain acyl-CoA intermediates and products of branched-chain amino acid metabolism. Kinetic characterization of the enzyme suggests a thigh regulation of the activity during different metabolic conditions in the mitochondria. Dietary ω-3 PUFAs from fish oil (FO) and krill oil (KO) cause different changes in lipid profiles and gene regulation when supplemented to mice. FO lowered most plasma lipids whereas KO only significantly lowered non-esterified fatty acids in plasma. FO showed a classical PPARα activation response by up regulating genes for fatty acid utilization and oxidation whereas KO down regulates genes for cholesterol and fatty acid synthesis

The mammalian peroxisome - "its function and distribution"

Peroxisomes are essential organelles for normal cell functions in all organisms from yeast to human. Their important contribution in different metabolic pathways is clearly visible by the severe phenotypes seen in the majority of peroxisomal diseases, where the symptoms often leads to an early death. Peroxisomes are involved in the synthesis of etherphospholipids and bile acids, in the metabolism of certain amino acids, purines and glyoxylate, and do also harbour an advanced system for degradation of various types of fatty acids and complex lipids. Peroxisomes are dynamic organelles that respond to different physiological and pharmacological changes by changing their number and contents of certain proteins. We carried out a tissue expression and regulation study on the majority of all known peroxisomal proteins (here called the ‘Pexiome’) in mouse at mRNA level to investigate if and how the different pathways may differ in their expression through out the mouse body. We studied how the mRNA expression varies in liver, kidney and intestinal epithelial in response to 12 hrs fasting, and also the effect of peroxisome proliferator activating receptor α (PPARα) agonist administration on gene expression in liver. The results show that indeed the mRNA expression of different genes varies markedly among tissues, while a number of genes seem to have a very wide tissue expression, which is in line with the content of peroxisomes in all cell types. Interestingly, fasting has a profound effect on the expression of the ‘Pexiome’ and also affects the peroxisomal gene expression in a strongly tissue specific manner. By examination of mouse livers from fasted and PPARα agonist treated animals on PPARα (+/+) and PPARα (-/-) backgrounds, it was evident that the regulation of most of the peroxisomal genes by fasting is far more complex than just involving PPARα activation. We also carried out an in depth study on the mouse peroxisomal Nudix hydrolase 7α (NUDT7α), which had previously been shown to act as a CoASH diphosphatase. Our data show that NUDT7α preferably cleaves off 3’,5’-ADP from the CoA-moiety of medium chain acyl-CoA’s. The expression of the enzyme at mRNA level was down regulated during PPARα activation in liver, and we also found that the total Nudix hydrolase activity was decreased in rat liver peroxisomes isolated from clofibrate treated mice. These findings suggest that NUDT7α may be an important regulator of the peroxisomal CoASH pool, and likely also regulates the β-oxidation of fatty acids in the peroxisome at substrate leve

Fish oil and krill oil supplementations differentially regulate lipid catabolic and synthetic pathways in mice

Background: Marine derived oils are rich in long-chain polyunsaturated omega-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have long been associated with health promoting effects such as reduced plasma lipid levels and anti-inflammatory effects. Krill oil (KO) is a novel marine oil on the market and is also rich in EPA and DHA, but the fatty acids are incorporated mainly into phospholipids (PLs) rather than triacylglycerols (TAG). This study compares the effects of fish oil (FO) and KO on gene regulation that influences plasma and liver lipids in a high fat diet mouse model. Methods: Male C57BL/6J mice were fed either a high-fat diet (HF) containing 24% (wt/wt) fat (21.3% lard and 2.3% soy oil), or the HF diet supplemented with FO (15.7% lard, 2.3% soy oil and 5.8% FO) or KO (15.6% lard, 2.3% soy oil and 5.7% KO) for 6 weeks. Total levels of cholesterol, TAG, PLs, and fatty acid composition were measured in plasma and liver. Gene regulation was investigated using quantitative PCR in liver and intestinal epithelium. Results: Plasma cholesterol (esterified and unesterified), TAG and PLs were significantly decreased with FO. Analysis of the plasma lipoprotein particles indicated that the lipid lowering effect by FO is at least in part due to decreased very low density lipoprotein (VLDL) content in plasma with subsequent liver lipid accumulation. KO lowered plasma non-esterified fatty acids (NEFA) with a minor effect on fatty acid accumulation in the liver. In spite of a lower omega-3 fatty acid content in the KO supplemented diet, plasma and liver PLs omega-3 levels were similar in the two groups, indicating a higher bioavailability of omega-3 fatty acids from KO. KO more efficiently decreased arachidonic acid and its elongation/desaturation products in plasma and liver. FO mainly increased the expression of several genes involved in fatty acid metabolism, while KO specifically decreased the expression of genes involved in the early steps of isoprenoid/ cholesterol and lipid synthesis. Conclusions: The data show that both FO and KO promote lowering of plasma lipids and regulate lipid homeostasis, but with different efficiency and partially via different mechanisms

Plasma Cholesterol- and Body Fat-Lowering Effects of Chicken Protein Hydrolysate and Oil in High-Fat Fed Male Wistar Rats

Rest raw materials provide a new source of bioactive dietary ingredients, and this study aimed to determine the health effects of diets with chicken protein hydrolysate (CPH) and chicken oil (CO) generated from deboned chicken meat. Male Wistar rats (n = 56) were divided into seven groups in three predefined sub-experiments to study the effects of protein source (casein, chicken fillet, pork fillet, and CPH), the dose-effect of CPH (50% and 100% CPH), and the effects of combining CPH and CO. Rats were fed high-fat diets for 12 weeks, and casein and chicken fillet were used as controls in all sub-experiments. While casein, chicken-, or pork fillet diets resulted in similar weight gain and plasma lipid levels, the CPH diet reduced plasma total cholesterol. This effect was dose dependent and accompanied with the reduced hepatic activities of acetyl-CoA carboxylase and fatty acid synthase. Further, rats fed combined CPH and CO showed lower weight gain, and higher hepatic mitochondrial fatty acid oxidation, plasma L-carnitine, short-chain acylcarnitines, TMAO, and acetylcarnitine/palmitoylcarnitine. Thus, in male Wistar rats, CPH and CO lowered plasma cholesterol and increased hepatic fatty acid oxidation compared to whole protein diets, pointing to potential health-beneficial bioactive properties of these processed chicken rest raw materials.publishedVersio

Plasma Cholesterol- and Body Fat-Lowering Effects of Chicken Protein Hydrolysate and Oil in High-Fat Fed Male Wistar Rats

Rest raw materials provide a new source of bioactive dietary ingredients, and this study aimed to determine the health effects of diets with chicken protein hydrolysate (CPH) and chicken oil (CO) generated from deboned chicken meat. Male Wistar rats (n = 56) were divided into seven groups in three predefined sub-experiments to study the effects of protein source (casein, chicken fillet, pork fillet, and CPH), the dose-effect of CPH (50% and 100% CPH), and the effects of combining CPH and CO. Rats were fed high-fat diets for 12 weeks, and casein and chicken fillet were used as controls in all sub-experiments. While casein, chicken-, or pork fillet diets resulted in similar weight gain and plasma lipid levels, the CPH diet reduced plasma total cholesterol. This effect was dose dependent and accompanied with the reduced hepatic activities of acetyl-CoA carboxylase and fatty acid synthase. Further, rats fed combined CPH and CO showed lower weight gain, and higher hepatic mitochondrial fatty acid oxidation, plasma L-carnitine, short-chain acylcarnitines, TMAO, and acetylcarnitine/palmitoylcarnitine. Thus, in male Wistar rats, CPH and CO lowered plasma cholesterol and increased hepatic fatty acid oxidation compared to whole protein diets, pointing to potential health-beneficial bioactive properties of these processed chicken rest raw materials

Fish oil and krill oil differentially modify the liver and brain lipidome when fed to mice

Background: Marine food is an important source of omega-3 fatty acids with beneficial health effects. Oils from marine organisms have different fatty acid composition and differ in their molecular composition. Fish oil (FO) has a high content of eicosapentaenoic and docosahexaenoic acids mainly esterified to triacylglycerols, while in krill oil (KO) these fatty acids are mainly esterified to phospholipids. The aim was to study the effects of these oils on the lipid content and fatty acid distribution in the various lipid classes in liver and brain of mice. Methods: Mice were fed either a high-fat diet (HF), a HF diet supplemented with FO or with KO (n = 6). After six weeks of feeding, liver and brain lipid extracts were analysed using a shotgun and TAG lipidomics approach. Student t-test was performed after log-transformation to compare differences between study groups. Results: Six weeks of feeding resulted in significant changes in the relative abundance of many lipid classes compared to control mice. In both FO and KO fed mice, the triacylglycerol content in the liver was more than doubled. The fatty acid distribution was affected by the oils in both liver and brain with a decrease in the abundance of 18:2 and 20:4, and an increase in 20:5 and 22:6 in both study groups. 18:2 decreased in all lipid classes in the FO group but with only minor changes in the KO group. Differences between the feeding groups were particularly evident in some of the minor lipid classes that are associated with inflammation and insulin resistance. Ceramides and diacylglycerols were decreased and cholesteryl esters increased in the liver of the KO group, while plasmalogens were decreased in the FO group. In the brain, diacylglycerols were decreased, more by KO than FO, while ceramides and lactosylceramides were increased, more by FO than KO. Conclusion: The changes in the hepatic sphingolipids and 20:4 fatty acid levels were greater in the KO compared to the FO fed mice, and are consistent with a hypothesis that krill oil will have a stronger anti-inflammatory action and enhances insulin sensitivity more potently than fish oil

The PPAR pan-agonist tetradecylthioacetic acid promotes redistribution of plasma cholesterol towards large HDL

Tetradecylthioacetic acid (TTA) is a synthetic fatty acid with a sulfur substitution in the β-position. This modification renders TTA unable to undergo complete β-oxidation and increases its biological activity, including activation of peroxisome proliferator activated receptors (PPARs) with preference for PPARα. This study investigated the effects of TTA on lipid and lipoprotein metabolism in the intestine and liver of mice fed a high fat diet (HFD). Mice receiving HFD supplemented with 0.75% (w/w) TTA had significantly lower body weights compared to mice fed the diet without TTA. Plasma triacylglycerol (TAG) was reduced 3-fold with TTA treatment, concurrent with increase in liver TAG. Total cholesterol was unchanged in plasma and liver. However, TTA promoted a shift in the plasma lipoprotein fractions with an increase in larger HDL particles. Histological analysis of the small intestine revealed a reduced size of lipid droplets in enterocytes of TTA treated mice, accompanied by increased mRNA expression of fatty acid transporter genes. Expression of the cholesterol efflux pump Abca1 was induced in the small intestine, but not in the liver. Scd1 displayed markedly increased mRNA and protein expression in the intestine of the TTA group. It is concluded that TTA treatment of HFD fed mice leads to increased expression of genes involved in uptake and transport of fatty acids and HDL cholesterol in the small intestine with concomitant changes in the plasma profile of smaller lipoproteins