Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma.

Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA beta-oxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.

L-carnitine exerts a nutrigenomic effect via direct modulation of nuclear receptor signaling in adipocytes, hepatocytes and SKMC, demonstrating its nutritional impact.

L-carnitine is an indispensable metabolite facilitating the transport of fatty acids into the mitochondrial matrix and has been previously postulated to exert a nutrigenomic effect. However, the underlying molecular mechanisms remain mostly unclear. We hypothesized that L-carnitine interacts with nuclear receptors involved in metabolic regulation, thereby modulating downstream targets of cellular metabolism. Therefore, we investigated the effect of L-carnitine supplementation on protein activity, mRNA expression, and binding affinities of nuclear receptors as well as mRNA expression of downstream targets in skeletal muscle cells, hepatocytes, and differentiated adipocytes. L-carnitine supplementation to hepatocytes increased the protein activity of multiple nuclear receptors (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). Diverging effects on the mRNA expression of PPAR-α, PPAR-δ, PPAR-γ, RAR-ß, LXR-α, and RXR-α were observed in adipocytes, hepatocytes, and skeletal muscle cells. mRNA levels of PPAR-α, a key regulator of lipolysis and ß-oxidation, were significantly upregulated, emphasizing a role of L-carnitine as a promoter of lipid catabolism. L-carnitine administration to hepatocytes modulated the transcription of key nuclear receptor target genes, including ALDH1A1, a promoter of adipogenesis, and OGT, a contributor to insulin resistance. Electrophoretic mobility shift assays proved L-carnitine to increase binding affinities of nuclear receptors to their promoter target sequences, suggesting a molecular mechanism for the observed transcriptional modulation. Overall, these findings indicate that L-carnitine modulates the activity and expression of nuclear receptors, thereby promoting lipolytic gene expression and decreasing transcription of target genes linked to adipogenesis and insulin resistance.

Diosgenin ameliorates palmitic acid-induced lipid accumulation via AMPK/ACC/CPT-1A and SREBP-1c/FAS signaling pathways in LO2 cells.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is characterized by excessive hepatic lipid accumulation. Many studies have suggested that lipid overload is the key initial factor that contributes to hepatic steatosis. Our previous study indicated that diosgenin (DSG) has a beneficial effect on energy metabolism, but the underlying mechanism remains unclear. METHODS: Human normal hepatocytes (LO2 cells) were incubated with palmitic acid to establish the cell model of nonalcoholic fatty liver. The effects of DSG on lipid metabolism, glucose uptake and mitochondrial function were evaluated. Furthermore, the mechanism of DSG on oxidative stress, lipid consumption and lipid synthesis in LO2 cells was investigated. RESULTS: The results indicated that palmitic acid induced obvious lipid accumulation in LO2 cells and that DSG treatment significantly reduced the intracellular lipid content. DSG treatment upregulated expression of lipolysis proteins, including phospho-AMP activated protein kinase (p-AMPK), phospho-acetyl-coA carboxylase (p-ACC) and carnitine acyl transferase 1A (CPT-1A), and inhibited expression of lipid synthesis-related proteins, including sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS). Additionally, DSG-treated cells displayed a marked improvement in mitochondrial function, with less production of reactive oxygen species and a higher mitochondrial membrane potential compared with the model group. CONCLUSION: This study suggests that DSG can reduce intracellular lipid accumulation in LO2 cells and that the underlying mechanism may be related to the improving oxidative stress, increasing fatty acid ß-oxidation and decreasing lipid synthesis. The above changes might be mediated by the activation of the AMPK/ACC/CPT-1A pathway and inhibition of the SREBP-1c/FAS pathway.

l-carnitine supplementation during vitrification or warming of in vivo-produced ovine embryos does not affect embryonic survival rates, but alters CrAT and PRDX1 expression.

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

Studies on the chemopreventive effect of carnitine on tumorigenesis in vivo, using two experimental murine models of colon cancer.

Carnitine is known for its essential role in intermediary metabolism. In vitro studies suggest that its antioxidant and anti-inflammatory properties are potentially beneficial toward cancer prevention. This study tested effects of carnitine on the development of colon cancer in vivo using 2 murine models: azoxymethane (AOM) treatment as a model of carcinogen-induced colon cancer and a genetically induced model using Apc (Min/+) mice. AOM and Apc (Min/+) mice divided into dietary groups varying in lipid content, with or without carnitine supplementation (0.08%). AOM-exposed mice on a high butterfat diet had significantly increased aberrant crypts (ACF) (9.3 ± 0.88 vs. 6.3 ± 0.65), and macroscopic tumors (3.8 ± 0.95 vs. 2.0 ± 0.25) compared to mice on a control diet. In AOM mice fed the high butterfat diet, carnitine supplementation inhibited ACF (4.9 ± 0.7 vs. 9.3 ± 0.88, P < 0.001), crypt multiciplicity (1.6 ± 0.08 vs. 1.92 ± 0.1, P < 0.01) and tumors (1.5 ± 0.38 vs. 3.8 ± 0.95, P < 0.001). Carnitine supplementation resulted in significantly increased tissue carnitine and acylcarnitine levels. Carnitine inhibited the development of precancerous lesions and macroscopic colonic tumors in AOM-treated mice. However, carnitine did not exert protective effects on intestinal tumors in Apc (Min/+) mice.

Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

Age-associated mitochondrial oxidative decay: improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L- carnitine and/or R-alpha -lipoic acid.

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-l-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-alpha-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

Dietary l-carnitine stimulates carnitine acyltransferases in the liver of aged rats.

Aging affects oxidative metabolism in liver and other tissues. Carnitine acyltransferases are key enzymes of this process in mitochondria. As previously shown, the rate of transcription and activity of carnitine palmitoyltransferase CPT1 are also related to carnitine levels. In this study we compared the effect of dietary l-carnitine (100 mg l-carnitine/kg body weight/day over 3 months) on liver enzymes of aged rats (months 21-24) to adult animals (months 6-9) and age-related controls for both groups. The transcription rate of CPT1, CPT2, and carnitine acetyltransferase (CRAT) was determined by quantitative reverse transcription real-time PCR (RTQPCR) and compared to the activity of the CPT1A enzyme. The results showed that the transcription rates of CPT1, CPT2, and CRAT were similar in aged and adult control animals. Carnitine-fed old rats had a significant (p<0.05) 8-12-fold higher mean transcription rate of CPT1 and CRAT compared to aged controls, adult carnitine-fed animals, and adult controls, whereas the transcription rate of CPT2 was stimulated 2-3-fold in carnitine-fed animals of both age groups. With regard to the enzymatic activity of CPT1 there was a 1.5-fold increase in the old carnitine group compared to all other groups. RNA in situ hybridization also indicated an enhanced expression of CPT1A in hepatocytes from l-carnitine-supplemented animals. These results suggest that l-carnitine stimulates transcription of CPT1, CPT2, and CRAT as well as the enzyme activity of CPT1 in the livers of aged rats.

Expression of muscarinic receptor types in the primate ovary and evidence for nonneuronal acetylcholine synthesis.

The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.

Effect of dehydroepiandrosterone sulfate on carnitine acetyl transferase activity and L-carnitine levels in oophorectomized rats.

Alteration in energy metabolism of postmenopausal women might be related to the reduction of dehydroepiandrosterone sulfate (DHEAS). DHEA and DHEAS decline with age, leveling at their nadir near menopause. DHEA and DHEAS modulate fatty acid metabolism by regulating carnitine acyltransferases and CoA. The purpose of this study was to determine whether dietary supplementation with DHEAS would also increase tissue L-carnitine levels, carnitine acetyltransferase (CAT) activity and mitochondrial respiration in oophorectomized rats. Plasma L-carnitine levels rose following oophorectomy in all groups (P < 0.0001). Supplementation with DHEAS was not associated with further elevation of plasma L-carnitine levels, but with increased hepatic total and free L-carnitine (P = 0.021 and P < 0.0001, respectively) and cardiac total L-carnitine concentrations (P = 0.045). In addition, DHEAS supplementation increased both hepatic and cardiac CAT activities (P < 0.0001 and P = 0.05 respectively). CAT activity positively correlated with the total and free carnitine levels in both liver and heart (r = 0.764, r = 0.785 and r = 0.700, r = 0.519, respectively). Liver mitochondrial respiratory control ratio, ADP:O ratio and oxygen uptake were similar in both control and supplemented groups. These results demonstrate that in oophorectomized rats, dietary DHEAS supplementation increases the liver and heart L-carnitine levels and CAT activities. In conclusion, DHEAS may modulate L-carnitine level and CAT activity in estrogen deficient rats. The potential role of DHEAS in the regulation of fatty acid oxidation in postmenopausal women is worthy of investigation.

Inhibition instead of enhancement of lipid peroxidation by pretreatment with the carcinogenic peroxisome proliferator nafenopin in rat liver exposed to a high single dose of corn oil.

Oxidative stress is discussed as a possible hepatocarcinogenic mechanism of peroxisome proliferators (PP) in rodents and is suggested to result from the induction of peroxisomal beta-oxidation (PBOX) by PP. The induced PBOX is assumed to produce excessive H2O2 from the degradation of fatty acids, ultimately leading to oxidative stress and lipid peroxidation. In the present short term-study, we attempted to stimulate lipid peroxidation in male Wistar rats by (1) inducing PBOX enzymes with the peroxisome proliferator nafenopin at 90 mg/kg body weight per day in the diet for 10-11 days, and (2) by supplying the induced PBOX with an abundant amount of fatty acid as substrate, using a corn oil gavage at 20 ml/kg body weight. The corn-oil gavage alone, i.e. without preceding nafenopin treatment, enhanced liver triacylglycerol nine- to tenfold and hepatic lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), was increased 50% compared with controls. Both observations were made after 18 h when the peak elevations occurred. Upon pretreatment with nafenopin, associated with a sevenfold induction of PBOX, the corn oil gavage however caused only a threefold maximal increase in hepatic triacylglycerol, also at the 18 h time-point; TBARS remained almost at control levels, as monitored at seven time points over 24-25 h. These results suggest that nafenopin reduces rather than enhances lipid peroxidation, despite the provision, in a short term study, of high doses of substrate to the induced enzyme system that is hypothetically causing oxidative stress in the liver.

Muscle carnitine deficiency in patients with severe peripheral vascular disease.

BACKGROUND: This study was designed to evaluate the effect of severe peripheral arterial insufficiency on carnitine concentrations and carnitine acetyltransferase and palmitoyltransferase activities in the ischemic skeletal muscles of patients with severe peripheral vascular disease. METHODS AND RESULTS: Nine biopsy specimens of ischemic muscles were obtained from five patients undergoing reconstructive vascular surgery. Biopsies from 35 normal subjects served as controls. Ischemic muscles showed a significant reduction in total carnitine from the control value of 20.9 +/- 5.2 to 11.6 +/- 6.2 nmol/mg noncollagen protein (p less than 0.01). A significantly lower free carnitine and acylcarnitine content contributed to this reduction. Similarly, carnitine acetyltransferase activity was reduced in the ischemic muscles from the control value of 102.1 +/- 41.2 to 52.9 +/- 22.1 nmol/min/mg noncollagen protein (p less than 0.01). On the contrary, carnitine palmitoyltransferase activity did not show any change (0.29 +/- 0.05 nmol/min/mg noncollagen protein in the ischemic muscles and 0.28 +/- 0.07 nmol/min/mg noncollagen protein in controls). Carnitine, acylcarnitines, and enzyme activities were also measured in the ischemic muscles in four additional patients 2 days after intravenous administration of L-propionylcarnitine (1.5 g as a single bolus followed by an infusion of 1 mg/kg/min for 30 minutes). Treatment restored normal levels of carnitine and its esters in the ischemic muscles but did not affect enzyme activities. CONCLUSIONS: Demonstration of carnitine deficiency in severe peripheral vascular disease substantiates previous findings showing the efficacy of carnitine supplementation to ischemic muscles. Furthermore, the feasibility of restoring carnitine homeostasis with L-propionylcarnitine provides the basis for clinical trials aimed at assessing the efficacy of this carnitine ester in the treatment of peripheral vascular disease.

Species and strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids.

B6C3F1 mice and Sprague-Dawley rats were provided drinking water containing 6-31 mM (1-5 g/liter) trichloroacetic acid (TCA), 8-39 mM (1-5 g/liter) dichloroacetic acid (DCA), or 11-32 mM (1-3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxisome proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased rat liver peroxisome proliferation. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osborne-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osborne-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement of liver peroxisome proliferation by TCA and DCA and suggest that if peroxisome proliferation is critical for the induction of hepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction.

Carnitine supplementation and depletion: tissue carnitines and enzymes in fatty acid oxidation.

Sixty-two male rats were randomly assigned into a 3 X 2 X 2 factorial design containing 12 groups according to carnitine treatment, exercise training (treadmill, 1 h, 5 times/wk, 8 wk, 26.8 m/min, 15% grade), and physical activity [rested for 60 h before they were killed or with an acute bout of exercise (1 h, 26.8 m/min, 15% grade) immediately before they were killed]. Isotonic saline was injected intraperitoneally 5 times/wk in the controls, whereas 750 mg/kg of L- or D-carnitine, respectively, were injected in the supplemented and depleted treatment groups. A significant increase in free and short-chain acyl carnitine concentration in skeletal muscle and heart was observed in L-carnitine supplemented rats, whereas a significant reduction in skeletal muscle, heart, and liver occurred in rats depleted of L-carnitine. Long-chain acyl carnitine in all tissues was not altered by carnitine treatment; training increased plasma and liver concentrations, whereas acute exercise decreased skeletal muscle and increased liver concentrations. An acute bout of exercise significantly increased short-chain acylcarnitine in liver, regardless of carnitine and/or training effects. beta-Hydroxyacyl-CoA dehydrogenase activity in skeletal muscle was induced by training but reduced by depletion. Carnitine acetyltransferase (CAT) was significantly increased in heart by L-carnitine supplementation, whereas it was reduced by depletion in skeletal muscle. Exercise training significantly increased CAT activity in skeletal muscle but not in heart, whereas acute exercise significantly increased activity in both tissues. Carnitine palmitoyltransferase activity was increased by acute exercise in the heart in only the supplemented and exercise-trained rats.

Concentration-dependent inhibition of development of GGT positive foci in rat liver by the environmental contaminant di(2-ethylhexyl) phthalate.

The ability of di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer and environmental contaminant, to suppress development of putative preneoplastic lesions in rat liver was evaluated. gamma-Glutamyl transpeptidase-positive (GGT+) foci were initiated in the livers of Sprague-Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. Promotion of foci was commenced by feeding a choline-deficient diet (CD). A group of control rats was fed a choline-supplemented diet (CS). The ability of DEHP to suppress the emergence of GGT+ foci was evaluated by feeding additional groups of rats the CD diet containing either 0.1%, 0.5%, 1.0% or 2.0% DEHP. The CD diet promoted the number of GGT+ foci above levels in control livers. Inclusion of the plasticizer to the levels of 0.5%, 1.0% and 2.0% in the CD diet effectively inhibited the appearance of the foci. However, DEHP was unable to inhibit the promoting effect of the CD diet at a concentration of 0.1%. DEHP's ability to block development of GGT+ foci correlated with its ability to increase liver weight and to induce carnitine acetyltransferase (EC 2.3.1.7), a marker of peroxisome proliferation.

Effect of fenofibrate and LF 2151 on hepatic peroxisomes in hamsters.

Hamsters were given a diet containing fenofibrate (0.5% or 0.05%) or its metabolite, LF 2151 (0.15% or 0.015%) or a standard diet for a 3-week period. At the end of this period, the analysis of plasma lipids showed that the mean plasma triglyceride concentrations were not significantly different in the five groups of animals. The mean plasma cholesterol concentrations were significantly reduced in animals treated with both drugs but only when given at the high dosage. No consistent changes were noted in the liver weight/body weight ratio and the DNA content of the liver; the number of peroxisomes was increased in the hepatocytes of animals given fenofibrate at the high dosage. Liver homogenates were fractionated and the fractions rich in peroxisomes were used for assays of several enzymes involved in lipid metabolism. Compared with the control animals, activity of cyanide-insensitive fatty acyl-CoA (FA-CoA) oxidizing system was significantly increased by fenofibrate at the high dosage, carnitine acetyltransferase activity was markedly increased by both drugs at the high dosage and catalase activity remained unmodified. As there was a significant inverse correlation between the peroxisomal activity of FA-CoA oxidizing system and the plasma cholesterol concentrations, it is suggested that the increase of peroxisomal beta-oxidation activity can be involved in the hypocholesterolemic action of fenofibrate and LF 2151. This is further substantiated by the finding that fenofibrate and LF 2151 were present in the peroxisomal fraction only in hamsters displaying hypocholesterolemia and high activity of FA-CoA oxidizing system. The presence of fenofibric acid in the plasma of hamsters given LF 2151 suggested that hepatocytes are able to generate the parent drug from this metabolite, underlining that the pharmacokinetics of fenofibrate are rather complex in hamsters.

Computer simulation of metabolism in palmitate-perfused rat heart. I. Palmitate oxidation.

A computer model of the fatty acid oxidation pathway in perfused rat heart was constructed. It includes uptake, activation, and beta-oxidation of fatty acids, triglyceride synthesis and hydrolysis, and carnitine-dependent transport of acyl groups across the mitochondrial membrane under pseudosteady state conditions. Fatty acid utilization may be limited by beta-oxidation in hypoxia or ischemia but probably not in aerobic conditions. Nonesterified fatty acids bound to proteins are found to be metabolically available. The model predicts that stearate, but not palmitate, can support the highest observed respiration rate for perfused rat heart without supplementation by other substrates. Fatty acids are preferentially oxidized rather than being stored as triglycerides because the cystosolic acyl CoA level is lower than the Km for triglyceride synthesis. It is suggested that feedback inhibition of triglyceride lipase regulates utilization of triglycerides as fuel in aerobic hearts.