Changes in Plasma Pyruvate and TCA Cycle Metabolites upon Increased Hepatic Fatty Acid Oxidation and Ketogenesis in Male Wistar Rats.

Altered hepatic mitochondrial fatty acid ß-oxidation and associated tricarboxylic acid (TCA) cycle activity contributes to lifestyle-related diseases, and circulating biomarkers reflecting these changes could have disease prognostic value. This study aimed to determine hepatic and systemic changes in TCA-cycle-related metabolites upon the selective pharmacologic enhancement of mitochondrial fatty acid ß-oxidation in the liver, and to elucidate the mechanisms and potential markers of hepatic mitochondrial activity. Male Wistar rats were treated with 3-thia fatty acids (e.g., tetradecylthioacetic acid (TTA)), which target mitochondrial biogenesis, mitochondrial fatty acid ß-oxidation, and ketogenesis predominantly in the liver. Hepatic and plasma concentrations of TCA cycle intermediates and anaplerotic substrates (LC-MS/MS), plasma ketones (colorimetric assay), and acylcarnitines (HPLC-MS/MS), along with associated TCA-cycle-related gene expression (qPCR) and enzyme activities, were determined. TTA-induced hepatic fatty acid ß-oxidation resulted in an increased ratio of plasma ketone bodies/nonesterified fatty acid (NEFA), lower plasma malonyl-CoA levels, and a higher ratio of plasma acetylcarnitine/palmitoylcarnitine (C2/C16). These changes were associated with decreased hepatic and increased plasma pyruvate concentrations, and increased plasma concentrations of succinate, malate, and 2-hydroxyglutarate. Expression of several genes encoding TCA cycle enzymes and the malate-oxoglutarate carrier (Slc25a11), glutamate dehydrogenase (Gdh), and malic enzyme (Mdh1 and Mdh2) were significantly increased. In conclusion, the induction of hepatic mitochondrial fatty acid ß-oxidation by 3-thia fatty acids lowered hepatic pyruvate while increasing plasma pyruvate, as well as succinate, malate, and 2-hydroxyglutarate.

Sex-specific responses in glucose-insulin homeostasis and lipoprotein-lipid components after high-dose supplementation with marine n-3 PUFAs in abdominal obesity: a randomized double-blind crossover study.

Background: Clinical studies on effects of marine-derived omega-3 (n-3) polyunsaturated fatty acids (PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the plant-derived omega-6 (n-6) PUFA linoleic acid (LA) on lipoprotein-lipid components and glucose-insulin homeostasis have shown conflicting results, which may partly be explained by differential responses in females and males. However, we have lacked data on sexual dimorphism in the response of cardiometabolic risk markers following increased consumption of n-3 or n-6 PUFAs. Objective: To explore sex-specific responses after n-3 (EPA + DHA) or n-6 (LA) PUFA supplementation on circulating lipoprotein subfractions, standard lipids, apolipoproteins, fatty acids in red blood cell membranes, and markers of glycemic control/insulin sensitivity among people with abdominal obesity. Methods: This was a randomized double-blind crossover study with two 7-week intervention periods separated by a 9-week washout phase. Females (n = 16) were supplemented with 3 g/d of EPA + DHA (fish oil) or 15 g/d of LA (safflower oil), while males (n = 23) received a dose of 4 g/d of EPA + DHA or 20 g/d of LA. In fasting blood samples, we measured lipoprotein particle subclasses, standard lipids, apolipoproteins, fatty acid profiles, and markers of glycemic control/insulin sensitivity. Results: The between-sex difference in relative change scores was significant after n-3 for total high-density lipoproteins (females/males: -11%*/-3.3%, p = 0.036; *: significant within-sex change), high-density lipoprotein particle size (+2.1%*/-0.1%, p = 0.045), and arachidonic acid (-8.3%*/-12%*, p = 0.012), and after n-6 for total (+37%*/+2.1%, p = 0.041) and small very-low-density lipoproteins (+97%*/+14%, p = 0.021), and lipoprotein (a) (-16%*/+0.1%, p = 0.028). Circulating markers of glucose-insulin homeostasis differed significantly after n-3 for glucose (females/males: -2.1%/+3.9%*, p = 0.029), insulin (-31%*/+16%, p < 0.001), insulin C-peptide (-12%*/+13%*, p = 0.001), homeostasis model assessment of insulin resistance index 2 (-12%*/+14%*, p = 0.001) and insulin sensitivity index 2 (+14%*/-12%*, p = 0.001), and quantitative insulin sensitivity check index (+4.9%*/-3.4%*, p < 0.001). Conclusion: We found sex-specific responses after high-dose n-3 (but not n-6) supplementation in circulating markers of glycemic control/insulin sensitivity, which improved in females but worsened in males. This may partly be related to the sex differences we observed in several components of the lipoprotein-lipid profile following the n-3 intervention. Clinical trial registration: https://clinicaltrials.gov/, identifier [NCT02647333].

Changes in lipoprotein particle subclasses, standard lipids, and apolipoproteins after supplementation with n-3 or n-6 PUFAs in abdominal obesity: A randomized double-blind crossover study.

BACKGROUND & AIMS: Marine-derived omega-3 (n-3) polyunsaturated fatty acids (PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), lower circulating levels of triacylglycerols (TAGs), and the plant-derived omega-6 (n-6) PUFA linoleic acid (LA) may reduce cholesterol levels. Clinical studies on effects of these dietary or supplemental PUFAs on other blood fat fractions are few and have shown conflicting results. This study aimed to determine effects of high-dose supplemental n-3 (EPA + DHA) and n-6 (LA) PUFAs from high-quality oils on circulating lipoprotein subfractions and standard lipids (primary outcomes), as well as apolipoproteins, fatty acids, and glycemic control (secondary outcomes), in females and males with abdominal obesity. METHODS: This was a randomized double-blind crossover study with two 7-wk intervention periods separated by a 9-wk washout phase. Females (n = 16) were supplemented with 3 g/d of EPA + DHA (TAG fish oil) or 15 g/d of LA (safflower oil), while males (n = 23) received a dose of 4 g/d of EPA + DHA or 20 g/d of LA. In fasting blood samples, we investigated lipoprotein particle subclasses by nuclear magnetic resonance spectroscopy, as well as standard lipids, apolipoproteins, fatty acid profiles, and glucose and insulin. Data were analyzed by linear mixed-effects modeling with 'subjects' as the random factor. RESULTS: The difference between interventions in relative change scores was among the lipoprotein subfractions significant for total very-low-density lipoproteins (VLDLs) (n-3 vs. n-6: -38%∗ vs. +16%, p < 0.001; ∗: significant within-treatment change score), large VLDLs (-58%∗ vs. -0.91%, p < 0.001), small VLDLs (-57%∗ vs. +41%∗, p < 0.001), total low-density lipoproteins (LDLs) (+5.8%∗ vs. -4.3%∗, p = 0.002), large LDLs (+23%∗ vs. -2.1%, p = 0.004), total high-density lipoproteins (HDLs) (-6.0%∗ vs. +3.7%, p < 0.001), large HDLs (+11%∗ vs. -5.3%, p = 0.001), medium HDLs (-24%∗ vs. +6.2%, p = 0.030), and small HDLs (-9.9%∗ vs. +9.6%∗, p = 0.002), and among standard lipids for TAGs (-16%∗ vs. -2.6%, p = 0.014), non-esterified fatty acids (-19%∗ vs. +5.5%, p = 0.033), and total cholesterol (-0.28% vs. -4.4%∗, p = 0.042). A differential response in relative change scores was also found for apolipoprotein (apo)B (+0.40% vs. -6.0%∗, p = 0.008), apoA-II (-6.0%∗ vs. +1.5%, p = 0.001), apoC-II (-11%∗ vs. -1.7%, p = 0.025), and apoE (+3.3% vs. -3.8%, p = 0.028). CONCLUSIONS: High-dose supplementation of high-quality oils with n-3 (EPA + DHA) or n-6 (LA) PUFAs was followed by reductions in primarily TAG- or cholesterol-related markers, respectively. The responses after both interventions point to changes in the lipoprotein-lipid-apolipoprotein profile that have been associated with reduced cardiometabolic risk, also among people with TAG or LDL-C levels within the normal range. REGISTRATION: Registered under ClinicalTrials.gov Identifier: NCT02647333. CLINICAL TRIAL REGISTRATION: Registered at https://clinicaltrials.gov/ct2/show/NCT02647333.

Plasma 3-hydroxyisobutyrate (3-HIB) and methylmalonic acid (MMA) are markers of hepatic mitochondrial fatty acid oxidation in male Wistar rats.

OBJECTIVE: Discovery of specific markers that reflect altered hepatic fatty acid oxidation could help to detect an individual's risk of fatty liver, type 2 diabetes and cardiovascular disease at an early stage. Lipid and protein metabolism are intimately linked, but our understanding of this crosstalk remains limited. METHODS: In male Wistar rats, we used synthetic fatty acid analogues (3-thia fatty acids) as a tool to induce hepatic fatty acid oxidation and mitochondrial biogenesis, to gain new insight into the link between fatty acid oxidation, amino acid metabolism and TCA cycle-related intermediate metabolites in liver and plasma. RESULTS: Rats treated with 3-thia fatty acids had 3-fold higher hepatic, but not adipose and skeletal muscle, expression of the thioesterase 3-hydroxyisobutyryl-CoA hydrolase (Hibch), which controls the formation of 3-hydroxyisobutyrate (3-HIB) in the valine degradation pathway. Consequently, 3-thia fatty acid-stimulated hepatic fatty acid oxidation and ketogenesis was accompanied by decreased plasma 3-HIB and increased methylmalonic acid (MMA) concentrations further downstream in BCAA catabolism. The higher plasma MMA corresponded to higher MMA-CoA hydrolase activity and hepatic expression of GTP-specific succinyl-CoA synthase (Suclg2) and succinate dehydrogenase (Sdhb), and lower MMA-CoA mutase activity. Plasma 3-HIB correlated positively to plasma and hepatic concentrations of TAG, plasma total fatty acids, plasma NEFA and insulin/glucose ratio, while the reverse correlations were seen for MMA. CONCLUSION: Our study provides new insight into TCA cycle-related metabolic changes associated with altered hepatic fatty acid flux, and identifies 3-HIB and MMA as novel circulating markers reflective of mitochondrial ß-oxidation in male Wistar rats.

Hepatic steatosis induced in C57BL/6 mice by a non-ß oxidizable fatty acid analogue is associated with reduced plasma kynurenine metabolites and a modified hepatic NAD+/NADH ratio.

BACKGROUND: Non-alcoholic fatty liver disease is often associated with obesity, insulin resistance, dyslipidemia, and the metabolic syndrome in addition to mitochondrial dysfunction and nicotinamide adenine dinucleotide (NAD+) deficiency. The aim of this study was to investigate how inhibition of mitochondrial fatty acid oxidation using the compound tetradecylthiopropionic acid (TTP) would affect hepatic triacylglycerol level and plasma levels of kynurenine (Kyn) metabolites and nicotinamide. METHODS: 12 C57BL/6 mice were fed a control diet, or an intervention diet supplemented with 0.9% (w/w) tetradecylthiopropionic acid for 14 days. Blood and liver samples were collected, enzyme activities and gene expression were analyzed in liver, in addition to fatty acid composition. Metabolites in the tryptophan/kynurenine pathway and total antioxidant status were measured in plasma. RESULTS: Dietary treatment with tetradecylthiopropionic acid for 2 weeks induced fatty liver accompanied by decreased mitochondrial fatty acid oxidation. The liver content of the oxidized form of NAD+ was increased, as well as the ratio of NAD+/NADH, and these changes were associated by increased hepatic mRNA levels of NAD synthetase and nicotinamide mononucleotide adenyltransferase-3. The downstream metabolites of kynurenine were reduced in plasma whereas the plasma nicotinamide content was increased. Some effects on inflammation and oxidative stress was observed in the liver, while the plasma antioxidant capacity was increased. This was accompanied by a reduced plasma ratio of kynurenine/tryptophan. In addition, a significant decrease in the inflammation-related arachidonic fatty acid in liver was observed. CONCLUSION: Fatty liver induced by short-time treatment with tetradecylthiopropionic acid decreased the levels of kynurenine metabolites but increased the plasma levels of NAD+ and nicotinamide. These changes are most likely not associated with increased inflammation and oxidative stress. Most probably the increase of NAD+ and nicotinamide are generated through the Preiss Handler pathway and/or salvage pathway and not through the de novo pathway. The take home message is that non-alcoholic fatty liver disease is associated with the metabolic syndrome in addition to mitochondrial dysfunction and nicotinamide adenine dinucleotide (NAD+) deficiency. Inducing fatty liver in mice by inhibition of fatty acid oxidation resulted in a concomitant change in kynurenine metabolites increasing the plasma levels of nicotinamides and the hepatic NAD+/NADH ratio, probably without affecting the de novo pathway of kynurenines.

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 ß-position. This modification renders TTA unable to undergo complete ß-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.

Increased fatty acid oxidation and mitochondrial proliferation in liver are associated with increased plasma kynurenine metabolites and nicotinamide levels in normolipidemic and carnitine-depleted rats.

Dysregulation of the tryptophan (Trp)-NAD+ pathway has been related to several pathological conditions, and the metabolites in this pathway are known to influence mitochondrial respiration and redox status. The aim of this project was to investigate if stimulation of beta-oxidation and mitochondrial proliferation by the mitochondrial-targeted compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) would influence metabolites of the Trp-Kyn-NAD+ pathway. We wished to investigate how carnitine depletion by meldonium-treatment influenced these metabolites. After dietary treatment of male Wistar rats with 1-triple TTA for three weeks, increased hepatic mitochondrial- and peroxisomal fatty acid oxidation resulted. The plasma content of total carnitines decreased compared to control animals, whereas hepatic genes involved in CoA biosynthesis were upregulated by 1-triple TTA treatment. The plasma Trp level and individual metabolites in the kynurenine pathway were increased by 1-triple TTA, associated with decreased hepatic gene expression of indoleamine2,3-dioxygenase. 1-triple TTA treatment increased conversion of Trp to nicotinamide (Nam) as the plasma content of quinolinic acid, Nam and N1-methylnicotinamide (mNam) increased, accompanied with suppression of hepatic gene expression of α-amino-α-carboxymuconate-ε-semialdehyde decarboxylase. A positive correlation between mitochondrial fatty acid oxidation and Trp-derivatives was found. Almost identical results were obtained by 1-triple TTA in the presence of meldonium, which alone exerted minor effects. Moreover, the plasma Kyn:Trp ratio (KTR) correlated negatively to mitochondrial function. Whether increased flux through the Trp-NAD+ pathway increased redox status and lowered inflammation locally and systemically should be considered.

A mitochondria-targeted fatty acid analogue influences hepatic glucose metabolism and reduces the plasma insulin/glucose ratio in male Wistar rats.

A fatty acid analogue, 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), was previously shown to have hypolipidemic effects in rats by targeting mitochondrial activity predominantly in liver. This study aimed to determine if 1-triple TTA could influence carbohydrate metabolism. Male Wistar rats were treated for three weeks with oral supplementation of 100 mg/kg body weight 1-triple TTA. Blood glucose and insulin levels, and liver carbohydrate metabolism gene expression and enzyme activities were determined. In addition, human myotubes and Huh7 liver cells were treated with 1-triple TTA, and glucose and fatty acid oxidation were determined. The level of plasma insulin was significantly reduced in 1-triple TTA-treated rats, resulting in a 32% reduction in the insulin/glucose ratio. The hepatic glucose and glycogen levels were lowered by 22% and 49%, respectively, compared to control. This was accompanied by lower hepatic gene expression of phosphenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, and Hnf4A, a regulator of gluconeogenesis. Gene expression of pyruvate kinase, catalysing the final step of glycolysis, was also reduced by 1-triple TTA. In addition, pyruvate dehydrogenase activity was reduced, accompanied by 10-15-fold increased gene expression of its regulator pyruvate dehydrogenase kinase 4 compared to control, suggesting reduced entry of pyruvate into the TCA cycle. Indeed, the NADPH-generating enzyme malic enzyme 1 (ME1) catalysing production of pyruvate from malate, was increased 13-fold at the gene expression level. Despite the decreased glycogen level, genes involved in glycogen synthesis were not affected in livers of 1-triple TTA treated rats. In contrast, the pentose phosphate pathway seemed to be increased as the hepatic gene expression of glucose-6-phosphate dehydrogenase (G6PD) was higher in 1-triple TTA treated rats compared to controls. In human Huh7 liver cells, but not in myotubes, 1-triple-TTA reduced glucose oxidation and induced fatty acid oxidation, in line with previous observations of increased hepatic mitochondrial palmitoyl-CoA oxidation in rats. Importantly, this work recognizes the liver as an important organ in glucose homeostasis. The mitochondrially targeted fatty acid analogue 1-triple TTA seemed to lower hepatic glucose and glycogen levels by inhibition of gluconeogenesis. This was also linked to a reduction in glucose oxidation accompanied by reduced PHD activity and stimulation of ME1 and G6PD, favouring a shift from glucose- to fatty acid oxidation. The reduced plasma insulin/glucose ratio indicate that 1-triple TTA may improve glucose tolerance in rats.

Associations between fatty acid oxidation, hepatic mitochondrial function, and plasma acylcarnitine levels in mice.

BACKGROUND: The 4-thia fatty acid tetradecylthiopropionic acid (TTP) is known to inhibit mitochondrial ß-oxidation, and can be used as chemically induced hepatic steatosis-model in rodents, while 3-thia fatty acid tetradecylthioacetic acid (TTA) stimulates fatty acid oxidation through activation of peroxisome proliferator activated receptor alpha (PPARα). We wished to determine how these two compounds affected in vivo respiration and mitochondrial efficiency, with an additional goal to elucidate whether mitochondrial function is reflected in plasma acylcarnitine levels. METHODS: C57BL/6 mice were divided in 4 groups of 10 mice and fed a control low-fat diet, low-fat diets with 0.4% (w/w) TTP, 0.4% TTA or a combination of these two fatty acids for three weeks (n = 10). At sacrifice, ß-oxidation and oxidative phosphorylation (OXPHOS) capacity was analysed in fresh liver samples. Hepatic mitochondria were studied using transmission electron microscopy. Lipid classes were measured in plasma, heart and liver, acylcarnitines were measured in plasma, and gene expression was measured in liver. RESULTS: The TTP diet resulted in hepatic lipid accumulation, plasma L-carnitine and acetylcarnitine depletion and elevated palmitoylcarnitine and non-esterified fatty acid levels. No significant lipid accumulation was observed in heart. The TTA supplement resulted in enhanced hepatic ß-oxidation, accompanied by an increased level of acetylcarnitine and palmitoylcarnitine in plasma. Analysis of mitochondrial respiration showed that TTP reduced oxidative phosphorylation, while TTA increased the maximum respiratory capacity of the electron transport system. Combined treatment with TTP and TTA resulted in a profound stimulation of genes involved in the PPAR-response and L-carnitine metabolism, and partly prevented triacylglycerol accumulation in the liver concomitant with increased peroxisomal ß-oxidation and depletion of plasma acetylcarnitines. Despite an increased number of mitochondria in the liver of TTA + TTP fed mice, the OXPHOS capacity was significantly reduced. CONCLUSION: This study indicates that fatty acid ß-oxidation directly affects mitochondrial respiratory capacity in liver. As plasma acylcarnitines reflected the reduced mitochondrial ß-oxidation in TTP-fed mice, they could be useful tools to monitor mitochondrial function. As mitochondrial dysfunction is a major determinant of metabolic disease, this supports their use as plasma markers of cardiovascular risk in humans. Results however indicate that high PPAR activation obscures the interpretation of plasma acylcarnitine levels.

Chicken Protein Hydrolysates Have Anti-Inflammatory Effects on High-Fat Diet Induced Obesity in Mice.

Background: Studies have shown that dietary source of protein and peptides can affect energy metabolism and influence obesity-associated diseases. This study aimed to investigate the impact of different chicken protein hydrolysates (CPHs) generated from chicken rest raw materials in a mouse obesity model. Methods: Male C57BL/6 mice were fed a high-fat, high-sucrose diet with casein or CPHs generated using Papain + Bromelain, Alcalase, Corolase PP, or Protamex for 12 weeks (n = 12). Body weight, feed intake, and intraperitoneal glucose tolerance was determined, and plasma and liver and adipose tissues were collected at sacrifice. Results: The average feed intake and body weight did not differ between the groups and white adipose tissue depots were unchanged, except for a reduction in the subcutaneous depot in mice fed the Protamex CPH diet. Moreover, the CPH diets did not prevent increased fasting glucose and insulin levels. Interestingly, the hepatic mitochondrial fatty acid ß-oxidation was increased in mice fed Alcalase and Corolase PP CPHs. All CPH diets reduced plasma interleukine (IL)-1ß, interferon-γ, tumor necrosis factor α, and monocyte chemotactic protein 1 compared to control, indicating anti-inflammatory effects. In addition, Corolase PP and Protamex CPHs significantly reduced plasma levels of IL-1α, IL-2, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor. Conclusions: CPH diets were not able to counteract obesity and glucose intolerance in a mouse obesity model, but strongly reduced inflammatory parameters associated with obesity. Alcalase and Corolase PP CPHs also stimulated mitochondrial fatty acid ß-oxidation. The possibility that hydrolysates from chicken rest raw materials could alleviate obesity-associated metabolic disease should be investigated further.

The molecular structure of thio-ether fatty acids influences PPAR-dependent regulation of lipid metabolism.

Thio-ether fatty acids (THEFAs), including the parent 2-(tetradecylthio)acetic acid (TTA), are modified fatty acids (FAs) that have profound effects on lipid metabolism given that they are blocked for ß-oxidation, and able to act as peroxisome proliferator-activated receptor (PPAR) agonists. Therefore, TTA in particular has been tested clinically for its therapeutic potential against metabolic syndrome related disorders. Here, we describe the preparation of THEFAs based on the TTA scaffold with either a double or a triple bond. These are tested in cultured human skeletal muscle cells (myotubes), either as free acid or following esterification as phospholipids, lysophospholipids or monoacylglycerols. Metabolic effects are assessed in terms of cellular bioavailabilities in myotubes, by FA substrate uptake and oxidation studies, and gene regulation studies with selected PPAR-regulated genes. We note that the inclusion of a triple bond promotes THEFA-mediated FA oxidation. Furthermore, esterification of THEFAs as lysophospholipids also promotes FA oxidation effects. Given that the apparent clinical benefits of TTA administration were offset by dose limitation and poor bioavailability, we discuss the possibility that a selection of our latest THEFAs and THEFA-containing lipids might be able to fulfill the therapeutic potential of the parent TTA while minimizing required doses for efficacy, side-effects and adverse reactions.

Tetradecylthiopropionic acid induces hepatic mitochondrial dysfunction and steatosis, accompanied by increased plasma homocysteine in mice.

BACKGROUND: Hepatic mitochondrial dysfunction plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Methyl donor supplementation has been shown to alleviate NAFLD, connecting the condition to the one-carbon metabolism. Thus, the objective was to investigate regulation of homocysteine (Hcy) and metabolites along the choline oxidation pathway during induction of hepatic steatosis by the fatty acid analogue tetradecylthiopropionic acid (TTP), an inhibitor of mitochondrial fatty acid oxidation. METHODS: Mice were fed a control diet, or diets containing 0.3 %, 0.6 %, or 0.9 % (w/w) TTP for 14 days. Blood and liver samples were collected, enzyme activities and gene expression were analyzed in liver, lipid and fatty acid composition in liver and plasma, one-carbon metabolites, B-vitamin status, carnitine and acylcarnitines were analyzed in plasma. RESULTS: Liver mitochondrial fatty acid oxidation decreased by 40 % and steatosis was induced in a dose dependent manner; total lipids increased 1.6-fold in animals treated with 0.3 % TTP, 2-fold with 0.6 % TTP and 2.1 fold with 0.9 % TTP compared to control. The higher hepatic concentration of fatty acids was associated with shortening of carbon-length. Furthermore, the inhibited fatty acid oxidation led to a 30-fold decrease in plasma carnitine and 9.3-fold decrease in acetylcarnitine at the highest dose of TTP, whereas an accumulation of palmitoylcarnitine resulted. Compared to the control diet, TTP administration was associated with elevated plasma total Hcy (control: 7.2 ± 0.3 umol/L, 0.9 % TTP: 30.5 ± 5.9 umol/L) and 1.4-1.6 fold increase in the one-carbon metabolites betaine, dimethylglycine, sarcosine and glycine, accompanied by changes in gene expression of the different B-vitamin dependent pathways of Hcy and choline metabolism. A positive correlation between total Hcy and hepatic triacylglycerol resulted. CONCLUSIONS: The TTP-induced inhibition of mitochondrial fatty acid oxidation was not associated with increased hepatic oxidative stress or inflammation. Our data suggest a link between mitochondrial dysfunction and the methylation processes within the one-carbon metabolism in mice.

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.

A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats.

The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP), with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.

Three differently generated salmon protein hydrolysates reveal opposite effects on hepatic lipid metabolism in mice fed a high-fat diet.

This study investigates the effects of salmon peptide fractions, generated using different enzymatic hydrolyzation methods, on hepatic lipid metabolism. Four groups of mice were fed a high-fat diet with 20% casein (control group) or 15% casein and 5% of peptide fractions (treatment groups E1, E2 and E4) for 6weeks. Weight gain was reduced in mice fed E1 and E4-diets compared to control, despite a similar feed intake. Reduced plasma and liver triacylglycerol levels in E1 and E4-mice were linked to reduced fatty acid synthase (FAS) activity and hepatic expression of lipogenic genes. By contrast, plasma and liver lipids increased in the E2 group, concomitant with increased hepatic FAS activity and Δ9 desaturase gene expression. Shotgun lipidomics showed that MUFAs were significantly reduced in the E1 and E4 groups, whereas PUFAs were increased, and the opposite was observed in the E2 group. In conclusion, bioactive peptides with distinctive properties could potentially be isolated from salmon hydrolysates.

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

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

High consumption of farmed salmon does not disrupt the steady state of persistent organic pollutants (POP) in human plasma and adipose tissue.

The aims of this study were to (1) determine any changes in the levels of persistent organic pollutants (POP) and mercury (Hg) in human plasma and adipose tissue and (2) examine associations between plasma levels of pollutants and dietary fat intake. Outpatients with different metabolic disorders (n = 42) consumed 380 g of farmed Atlantic salmon fillets or 60 g of salmon oil per week in two study periods of 15 wk each, and were compared with a control group (n = 14). Concentrations of POP and Hg were measured in salmon fillets, salmon oil capsules, plasma and abdominal fat biopsies from patients before and after intervention. Mean concentrations of hexachlorobenzene (HCB), p,p'-DDE, sum of indicator polychlorinated biphenyls (PCB) (id-PCB), and sum polybrominated diphenyl ethers (PBDE) in abdominal fat at intervention start were 21, 191, 267, and 4.2 ng/g lipid weight. After 15 or 30 wk of salmon consumption no significant changes in concentrations of POP and Hg in samples of human plasma and abdominal fat were observed, indicating that steady-state levels of these pollutants were not markedly affected. The lack of significant changes may partly be attributed to a limited number of samples, large interindividual variation in POP levels, and a large age span (20-70 yr). After adjusting for age, significant associations were found between different plasma long-chain fatty acid concentrations, including n-3 and n-6 fatty acids and oleic acid, and some of the POP. The results indicate that the latter have different food products as their main sources of human exposure.

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.

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

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

A fish protein hydrolysate alters fatty acid composition in liver and adipose tissue and increases plasma carnitine levels in a mouse model of chronic inflammation.

BACKGROUND: There is growing evidence that fish protein hydrolysate (FPH) diets affect mitochondrial fatty acid metabolism in animals. The aim of the study was to determine if FPH could influence fatty acid metabolism and inflammation in transgene mice expressing human tumor necrosis factor alpha (hTNFα). METHODS: hTNFα mice (C57BL/6 hTNFα) were given a high-fat (23%, w/w) diet containing 20% casein (control group) or 15% FPH and 5% casein (FPH group) for two weeks. After an overnight fast, blood, adipose tissue, and liver samples were collected. Gene expression and enzyme activity was analysed in liver, fatty acid composition was analyzed in liver and ovarian white adipose tissue, and inflammatory parameters, carnitine, and acylcarnitines were analyzed in plasma. RESULTS: The n-3/n-6 fatty acid ratio was higher in mice fed the FPH diet than in mice fed the control diet in both adipose tissue and liver, and the FPH diet affected the gene expression of ∆6 and ∆9 desaturases. Mice fed this diet also demonstrated lower hepatic activity of fatty acid synthase. Concomitantly, a lower plasma INF-γ level was observed. Plasma carnitine and the carnitine precursor γ-butyrobetaine was higher in the FPH-group compared to control, as was plasma short-chained and medium-chained acylcarnitine esters. The higher level of plasma acetylcarnitine may reflect a stimulated mitochondrial and peroxisomal ß-oxidation of fatty acids, as the hepatic activities of peroxisomal acyl-CoA oxidase 1 and mitochondrial carnitine palmitoyltransferase-II were higher in the FPH-fed mice. CONCLUSIONS: The FPH diet was shown to influence hepatic fatty acid metabolism and fatty acid composition. This indicates that effects on fatty acid metabolism are important for the bioactivity of protein hydrolysates of marine origin.