Robert Ringseis

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species.

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) alpha. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARalpha and are less responsive to treatment with PPARalpha agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARalpha target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARalpha activation in these tissues (P<.05). Fasted pigs had a higher activity of gamma-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARalpha activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.

Conjugated Linoleic Acid Isomers Reduce Cholesterol Accumulation in Acetylated LDL-Induced Mouse RAW264.7 Macrophage-Derived Foam Cells

Synthetic activators of peroxisome proliferator-activated receptors (PPAR)-α and -γ are capable of reducing macrophage foam cell cholesterol accumulation through the activation of genes involved in cholesterol homeostasis. Since conjugated linoleic acids (CLA) were also demonstrated to activate PPARα and PPARγ in vivo and in vitro, we tested the hypothesis that CLA are also capable of reducing macrophage foam cell cholesterol accumulation. Thus, mouse RAW264.7 macrophage-derived foam cells were treated with CLA isomers, c9t11-CLA and t10c12-CLA, and linoleic acid (LA), as reference fatty acid, and analyzed for the concentrations of free and esterified cholesterol, cholesterol efflux and expression of genes involved in cholesterol homeostasis (CD36, ABCA1, LXRα, NPC-1, and NPC-2). Treatment with c9t11-CLA and t10c12-CLA, but not LA, lowered cholesterol accumulation, stimulated acceptor-dependent cholesterol efflux, and increased relative mRNA concentrations of CD36, ABCA1, LXRα, NPC-1, and NPC-2 (Pxa0<xa00.05). In conclusion, the present study showed that CLA isomers reduce cholesterol accumulation in RAW264.7 macrophage-derived foam cells presumably by enhancing lipid acceptor-dependent cholesterol efflux.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.

Feeding oxidized fat during pregnancy up-regulates expression of PPARα-responsive genes in the liver of rat fetuses

BackgroundFeeding oxidized fats causes activation of peroxisome proliferator-activated receptor α (PPARα) in the liver of rats. However, whether feeding oxidized fat during pregnancy also results in activation of PPARα in fetal liver is unknown. Thus, this study aimed to explore whether feeding oxidized fat during pregnancy causes a PPARα response in fetal liver. Two experiments with pregnant rats which were administered three different diets (control; oxidized fat; clofibrate as positive control) in a controlled feeding regimen during either late pregnancy (first experiment) or whole pregnancy (second experiment) were performed.ResultsIn both experiments pregnant rats treated with oxidized fat or clofibrate had higher relative mRNA concentrations of the PPARα-responsive genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 (CYP4A1), L-type carnitin-palmitoyl transferase I (L-CPT I), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) in the liver than control rats (P < 0.05). In addition, in both experiments fetuses of the oxidized fat group and the clofibrate group also had markedly higher relative mRNA concentrations of ACO, CYP4A1, CPT I, MCAD, and LCAD in the liver than those of the control group (P < 0.05), whereas the relative mRNA concentrations of PPARα, SREBP-1c, and FAS did not differ between treatment groups. In the second experiment treatment with oxidized fat also reduced triacylglycerol concentrations in the livers of pregnant rats and fetuses (P < 0.05).ConclusionThe present study demonstrates for the first time that components of oxidized fat with PPARα activating potential are able to induce a PPARα response in the liver of fetuses. Moreover, the present study shows that feeding oxidized fat during whole pregnancy, but not during late pregnancy, lowers triacylglycerol concentrations in fetal livers.

Treatment with pharmacological peroxisome proliferator-activated receptor α agonist clofibrate increases intestinal carnitine absorption in rats

Activation of PPARalpha by clofibrate has recently been shown to cause upregulation of the high-affinity carnitine transporter novel organic cation transporter (OCTN) 2 in small intestine. This strongly suggests that PPARalpha activation in response to clofibrate treatment improves the absorption of carnitine from the diet. To test this hypothesis, we performed an experiment with rats which were fed diets with or without 5 g clofibrate/kg diet and with or without 5 g L-carnitine/kg diet. PPARalpha was significantly activated by clofibrate in small intestine as evidenced by increased relative mRNA concentrations of the PPARalpha target gene acyl-CoA oxidase (P < 0.05). Relative mRNA concentration of OCTN2 in small intestine was significantly increased by clofibrate (P < 0.05) but not the carnitine supplementation, whereas relative mRNA concentrations of other carnitine transporters (OCTN1, ATB(0+)) in small intestine were not influenced by either clofibrate or carnitine. The absorption rate of carnitine in small intestine was markedly higher in rats treated with clofibrate than in those treated without clofibrate (P < 0.05). In conclusion, the present study shows that administration of clofibrate to rats increases carnitine absorption in small intestine which is probably due to the observed upregulation of OCTN2 mediated by activation of PPARalpha.

Downregulation of peroxisome proliferator-activated receptor α and its coactivators in liver and skeletal muscle mediates the metabolic adaptations during lactation in mice

Previous studies have shown that genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis are downregulated in liver and skeletal muscle of rats during lactation. However, biochemical mechanisms underlying these important metabolic adaptations during lactation have not yet been elucidated. As all these genes are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (Pparalpha), we hypothesized that their downregulation is mediated by a suppression of Pparalpha during lactation. In order to investigate this hypothesis, we performed an experiment with lactating and nonlactating Pparalpha knockout and corresponding wild-type mice. In wild-type mice, lactation led to a considerable downregulation of Pparalpha, Ppar coactivators Pgc1alpha and Pgc1beta, and Pparalpha target genes involved in fatty acid uptake, fatty acid oxidation, and thermogenesis in liver and skeletal muscle (P<0.05). Pparalpha knockout mice had generally a lower expression of all these Pparalpha target genes in liver and skeletal muscle. However, in those mice, lactation did not lower the expression of genes involved in fatty acid utilization and thermogenesis in liver and skeletal muscle. Expression levels of Pparalpha target genes in lactating wild-type mice were similar than in lactating or nonlactating Pparalpha knockout mice. In conclusion, the present findings suggest that downregulation of Pparalpha and its coactivators in tissues with high rates of fatty acid catabolism is responsible for the reduced utilization of fatty acids in liver and skeletal muscle and the reduced thermogenesis occurring in the lactating animal, which aim to conserve energy and metabolic substrates for milk production in the mammary gland.

Supplementing Obese Zucker Rats with Niacin Induces the Transition of Glycolytic to Oxidative Skeletal Muscle Fibers

In the present study, we tested the hypothesis that niacin increases the oxidative capacity of muscle by increasing the oxidative type I muscle fiber content. Twenty-four obese Zucker rats were assigned to 2 groups of 12 rats that were fed either a control diet (O group) or a diet supplemented with 750 mg/kg diet niacin (O+N group) for 4 wk. In addition, one group of lean rats (L group) was included in the experiment and fed the control diet for 4 wk. Plasma and liver concentrations of TG were markedly greater in obese groups than in the L group but markedly lower in the O+N group than in the O group (P < 0.05). Rats of the O+N group had a higher percentage of oxidative type I fibers and higher mRNA levels of genes encoding regulators of muscle fiber composition (Ppard, Ppargc1a, Ppargc1b), angiogenic factors (Vegfa, Vegfb), and genes involved in fatty acid utilization (Cpt1b, Slc25a20, Slc22a4, Slc22a5, Slc27a1) and oxidative phosphorylation (Cox4i1, Cox6a2) and a higher activity of the mitochondrial oxidative enzyme succinate dehydrogenase in muscle than rats of the O and L groups (P < 0.05). These niacin-induced changes in muscle metabolic phenotype are indicative of an increased capacity of muscle for oxidative utilization of fatty acids and are likely mediated by the upregulation of Ppard, Ppargc1a, and Ppargc1b, which are key regulators of muscle fiber composition, mitochondrial biogenesis, angiogenesis, and genes involved in fatty acid catabolism and oxidative phosphorylation. The increased utilization of fatty acids by muscle might contribute to the strong TG-lowering effect of niacin treatment.

Conjugated linoleic acid isomers inhibit platelet-derived growth factor-induced NF-κB transactivation and collagen formation in human vascular smooth muscle cells

BackgroundAtherosclerosis is characterized by extensive thickening of the arterial intima partially resulting from deposition of collagen by vascular smooth muscle cells (SMCs). Polyunsaturated fatty acids stimulate collagen formation through NF-κB activation.Aim of the studyThe present study aimed to explore the effect of conjugated linoleic acids (CLAs) which are known to inhibit NF-κB activation on collagen formation by SMCs.MethodsVascular SMCs were cultured with 50xa0µmol/l of CLA isomers (c9t11-CLA, t10c12-CLA) or linoleic acid (LA) and analysed for collagen formation and NF-κB p50 transactivation.ResultsTreatment with CLA isomers but not LA significantly reduced PDGF-stimulated [3H] proline incorporation into cell layer protein of SMCs without altering cell proliferation. Simultaneous treatment with the PPARγ inhibitor T0070907 abrogated this effect. Treatment of SMCs with c9t11-CLA and t10c12-CLA significantly reduced PDGF-induced NF-κB p50 activation.ConclusionsCLA isomers inhibit PDGF-stimulated collagen production by vascular SMCs, which is considered to be a hallmark of atherosclerosis, in a PPARγ-dependent manner. Whether inhibition of the NF-κB-pathway is of significance for the reduction of collagen formation by CLA isomers needs further investigation.

Influence of pharmacological PPARα activators on carnitine homeostasis in proliferating and non-proliferating species

Former studies in rats demonstrated that starvation or treatment with the hypolipidemic drug clofibrate causes a marked increase in the concentration of carnitine in the liver. The molecular mechanisms underlying these phenomena in rats, however, have been largely unknown. Since both, fasting and clofibrate treatment lead to an activation of peroxisome proliferator-activated receptor alpha (PPARalpha), a ligand-activated transcription factor that acts as an important regulator of lipid metabolism and energy homeostasis, the hypothesis has been raised that activation of this nuclear receptor is responsible for the alterations in carnitine homeostasis observed in rodents by either stimulating carnitine uptake or carnitine biosynthesis or both of them. The present review summarizes recent evidence from studies with rodents and pigs supporting the hypothesis that activation of PPARalpha is responsible for the alterations in carnitine homeostasis previously observed. According to these novel results an essential role for PPARalpha in the regulation of carnitine uptake and carnitine biosynthesis in rodents and pigs has been clearly established. Due to the strong similarities in the gene response to PPARalpha agonists and the similar metabolic features and anatomic conditions between pigs and humans, it is likely that pharmacological PPARalpha agonists exert similar effects in humans.

Peroxisome proliferator–activated receptor α and enzymes of carnitine biosynthesis in the liver are down-regulated during lactation in rats

This study investigated the hypothesis that lactation lowers gene expression of peroxisome proliferator-activated receptor (PPAR) alpha in the liver and that this leads to a down-regulation of hepatic enzymes involved in carnitine synthesis and novel organic cation transporters (OCTNs). Thirty-two pregnant female rats were divided into 4 groups. In the first group, all pups were removed, whereas in the other groups, litters were adjusted to sizes of 4, 10, or 18 pups per dam. Dams suckling their litters, irrespective of litter size, had lower relative messenger RNA concentrations of PPARalpha, various classic PPARalpha target genes involved in fatty acid catabolism, as well as enzymes involved in carnitine synthesis (trimethyllysine dioxygenase, 4-N-trimethylaminobutyraldehyde dehydrogenase, gamma-butyrobetaine dioxygenase) and OCTN1 in the liver than dams whose litters were removed (P < .05). Moreover, dams suckling their litters had a reduced activity of gamma-butyrobetaine dioxygenase in the liver and reduced concentrations of carnitine in plasma, liver, and muscle compared with dams without litters (P < .05). In conclusion, the present study demonstrates for the first time that lactation leads to a down-regulation of PPARalpha and genes involved in hepatic carnitine synthesis and uptake of carnitine (OCTN1) in the liver, irrespective of litter size. It is moreover suggested that down-regulation of PPARalpha in the liver may be a means to conserve energy and metabolic substrates for milk production in the mammary gland.