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.

Hepatic Energy Metabolism Underlying Differential Lipidomic Responses to High-Carbohydrate and High-Fat Diets in Male Wistar Rats.

BACKGROUND: Low-carbohydrate diets are suggested to exert metabolic benefits by reducing circulating triacylglycerol (TG) concentrations, possibly by enhancing mitochondrial activity. OBJECTIVE: We aimed to elucidate mechanisms by which dietary carbohydrate and fat differentially affect hepatic and circulating TG, and how these mechanisms relate to fatty acid composition. METHODS: Six-week-old, ∼300 g male Wistar rats were fed a high-carbohydrate, low-fat [HC; 61.3% of energy (E%) carbohydrate] or a low-carbohydrate, high-fat (HF; 63.5 E% fat) diet for 4 wk. Parameters of lipid metabolism and mitochondrial function were measured in plasma and liver, with fatty acid composition (GC), high-energy phosphates (HPLC), carnitine metabolites (HPLC-MS/MS), and hepatic gene expression (qPCR) as main outcomes. RESULTS: In HC-fed rats, plasma TG was double and hepatic TG 27% of that in HF-fed rats. The proportion of oleic acid (18:1n-9) was 60% higher after HF vs. HC feeding while the proportion of palmitoleic acid (16:1n-7) and vaccenic acid (18:1n-7), and estimated activities of stearoyl-CoA desaturase, SCD-16 (16:1n-7/16:0), and de novo lipogenesis (16:0/18:2n-6) were 1.5-7.5-fold in HC vs. HF-fed rats. Accordingly, hepatic expression of fatty acid synthase (Fasn) and acetyl-CoA carboxylase (Acaca/Acc) was strongly upregulated after HC feeding, accompanied with 8-fold higher FAS activity and doubled ACC activity. There were no differences in expression of liver-specific biomarkers of mitochondrial biogenesis and activity (Cytc, Tfam, Cpt1, Cpt2, Ucp2, Hmgcs2); concentrations of ATP, AMP, and energy charge; plasma carnitine/acylcarnitine metabolites; or peroxisomal fatty acid oxidation. CONCLUSIONS: In male Wistar rats, dietary carbohydrate was converted into specific fatty acids via hepatic lipogenesis, contributing to higher plasma TG and total fatty acids compared with high-fat feeding. In contrast, the high-fat, low-carbohydrate feeding increased hepatic fatty acid content, without affecting hepatic mitochondrial fatty acid oxidation.

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.

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.

A fatty acid analogue targeting mitochondria exerts a plasma triacylglycerol lowering effect in rats with impaired carnitine biosynthesis.

L-carnitine is important for the catabolism of long-chain fatty acids in the mitochondria. We investigated how the triacylglycerol (TAG)-lowering drug 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) influenced lipid metabolism in carnitine-depleted, 3-(2,2,2-trimethylhydrazinium)propionate dehydrate (Mildronate; meldonium)-treated male Wistar rats. As indicated, carnitine biosynthesis was impaired by Mildronate. However, TAG levels of both plasma and liver were decreased by 1-triple TTA in Mildronate-treated animals. This was accompanied by increased gene expression of proteins involved in mitochondrial activity and proliferation and reduced mRNA levels of Dgat2, ApoB and ApoCIII in liver. The hepatic energy state was reduced in the group of Mildronate and 1-triple TTA as reflected by increased AMP/ATP ratio, reduced energy charge and induced gene expression of uncoupling proteins 2 and 3. The increase in mitochondrial fatty acid oxidation was observed despite low plasma carnitine levels, and was linked to strongly induced gene expression of carnitine acetyltransferase, translocase and carnitine transporter, suggesting an efficient carnitine turnover. The present data suggest that the plasma TAG-lowering effect of 1-triple TTA in Mildronate-treated rats is not only due to increased mitochondrial fatty acid oxidation reflected by increased mitochondrial biogenesis, but also to changes in plasma clearance and reduced TAG biosynthesis.

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.

Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats.

Hepatic mitochondrial function, APOC-III, and LPL are potential targets for triglyceride (TG)-lowering drugs. After 3 weeks of dietary treatment with the compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), the hepatic mitochondrial FA oxidation increased more than 5-fold in male Wistar rats. Gene expression analysis in liver showed significant downregulation of APOC-III and upregulation of LPL and the VLDL receptor. This led to lower hepatic (53%) and plasma (73%) TG levels. Concomitantly, liver-specific biomarkers related to mitochondrial biogenesis and function (mitochondrial DNA, citrate synthase activity, and cytochrome c and TFAM gene expression) were elevated. Interestingly, 1-triple TTA lowered plasma acetylcarnitine levels, whereas the concentration of ß-hydroxybutyrate was increased. The hepatic energy state was reduced in 1-triple TTA-treated rats, as reflected by increased AMP/ATP and decreased ATP/ADP ratios, whereas the energy state remained unchanged in muscle and heart. The 1-triple TTA administration induced gene expression of uncoupling protein (UCP)2 and UCP3 in liver. In conclusion, the 1-triple TTA-mediated clearance of blood TG may result from lowered APOC-III production, increased hepatic LPL gene expression, mitochondrial FA oxidation, and (re)uptake of VLDL facilitating drainage of FAs to the liver for ß-oxidation and production of ketone bodies as extrahepatic fuel. The possibility that UCP2 and UCP3 mediate a moderate degree of mitochondrial uncoupling should be considered.

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.

A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats.

Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC's content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis.