Carnitine Acyltransferase Activities Research Articles

Astilbin lowers the effective caffeine dose for decreasing lipid accumulation via activating AMPK in high‐fat diet‐induced obese mice

Caffeine has an anti-obesity effect, although chronic excessive caffeine consumption also causes caffeinism, which is marked by increased anxiety or depression, amongst other symptoms. The present study aimed to investigate whether the addition of flavonoids such as astilbin can reduce the caffeine dose needed to inhibit obesity. ICR mice (n = 80) were fed with normal diet, high-fat diet (HFD), HFD supplemented with astilbin, caffeine, or astilbin + caffeine for 12 weeks. When diets supplemented with astilbin, 0.3 g kg-1 diet caffeine had the same effect as 0.6 g kg-1 diet caffeine alone, and 0.6 g kg-1 diet caffeine combined with astilbin most effectively inhibited HFD-induced obesity. Astilbin improved the anti-obesity effects of caffeine on lipid accumulation via the activation of AMP-activated protein kinase α (AMPKα). (i) Activated AMPKα decreased lipid biosynthesis by suppressing the activity or mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase, sterol regulatory element binding protein 1c and its target gene fatty acid synthase. (ii) Activated AMPKα also up-regulated lipolysis by enhancing the expression of adipose triglyceride lipase and increasing the phosphorylation of hormone-sensitive lipase. (iii) Finally, activated AMPKα increased carnitine acyltransferase and acyl-CoA oxidase activities, which further promoted fatty acid β-oxidation. The results obtained in the present study indicate that astilbin may decrease the effective dose of caffeine needed for an anti-obesity effect and also suggest that it suppresses fat accumulation via the activation of AMPK. © 2020 Society of Chemical Industry.

Exogenous carnitine application augments transport of fatty acids into mitochondria and stimulates mitochondrial respiration in maize seedlings grown under normal and cold conditions

This study aimed to investigate whether exogenous application of carnitine stimulates transportation of fatty acids into mitochondria, which is an important part of fatty acid trafficking in cells, and mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions. Cold stress led to significant increases in lipase activity, which is responsible for the breakdown of triacylglycerols, and carnitine acyltransferase (carnitine acyltransferase I and II) activities, which are responsible for the transport of activated long-chain fatty acids into mitochondria. While exogenous application of carnitine has a similar promoting effect with cold stress on lipase activity, it resulted in further increases in the activity of carnitine acyltransferases compared to cold stress. The highest activity levels for these enzymes were recorded in the seedlings treated with cold plus carnitine. In addition, these increases were correlated with positive increases in the contents of free- and long-chain acylcarnitines (decanoyl-l-carnitine, lauroyl-l-carnitine, myristoyl-l-carnitine, and stearoyl-l-carnitine), and with decreases in the total lipid content. The highest values for free- and long-chain acylcarnitines and the lowest value for total lipid content were recorded in the seedlings treated with cold plus carnitine. On the other hand, carnitine with and without cold stress significantly upregulated the expression level of citrate synthase, which is responsible for catalysing the first reaction of the citric acid cycle, and cytochrome oxidase, which is the membrane-bound terminal enzyme in the electron transfer chain, as well as lipase. All these results revealed that on the one hand, carnitine enhanced transport of fatty acids into mitochondria by increasing the activities of lipase and carnitine acyltransferases, and, on the other hand, stimulated mitochondrial respiration in the leaves of maize seedlings grown under normal and cold conditions.

Metabolic Effects of Clenbuterol and Salbutamol on Pork Meat Studied Using Internal Extractive Electrospray Ionization Mass Spectrometry

Direct mass spectrometry analysis of metabolic effects of clenbuterol and salbutamol on pork quality at the molecular level is incredibly beneficial for food regulations, public health and the development of new anti-obesity drugs. With internal extractive electrospray ionization mass spectrometry (iEESI-MS), nutrients including creatine, amino acids, L-carnitine, vitamin B6, carnosine and phosphatidylcholines in pork tissue were identified, without sample pretreatment, using collision-induced dissociation (CID) experiments and by comparison with authentic compounds. Furthermore, normal pork samples were clearly differentiated from pork samples with clenbuterol and salbutamol via principal component analysis (PCA). Correlation analysis performed on the spectral data revealed that the above-mentioned nutrients strongly correlated with pork quality, and the absolute intensity of phosphatidylcholines in normal pork was much higher than pork contaminated by clenbuterol and salbutamol. Our findings suggested that clenbuterol and salbutamol may render effects on the activity of carnitine acyltransferase I, hence the process that L-carnitine transports long-chain fatty acids into mitochondria and the formation of phosphatidylcholines might be affected. However, the underlying metabolic mechanisms of clenbuterol and salbutamol on carnitine acyltransferase I requires more comprehensive studies in future work.

Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

Possible mechanism for species difference on the toxicity of pivalic acid between dogs and rats

In a high dose toxicity study of pivalic acid (PA), PA caused skeletal muscle disorder in dog, and a significant increase of pivaloyl carnitine (PC) was observed in canine muscle, but not in rat muscle. In order to understand species difference of the toxicity of PA, we compared the in vitro metabolism of PA among dog, rat and rabbit, especially focussing on the carnitine conjugate. Canine muscle showed low, but significant carnitine conjugating activity, while that of rat was negligible. Canine kidney mitochondria had significant activity in the pivaloyl CoA synthesis (7 nmol/mg protein/h), but muscle mitochondria showed only trace activity. Both kidney and muscle mitochondria displayed similar carnitine acyltransferase activity (2-3 nmol/mg protein/h) towards pivaloyl CoA. On the other hand, with respect to the activity of carnitine acyltransferase in the reverse direction using PC as substrate, canine muscle mitochondria showed higher activity than that of kidney mitochondria. This means that PC is not the final stable metabolite, but is converted easily to pivaloyl CoA in canine muscle. These results suggest one of the possible mechanisms for canine selective muscle disorder to be as follows. Only canine muscle can metabolize PA to its carnitine conjugate slowly, but significantly. In canine muscle, PC is not the final stable metabolite; it is easily converted to pivaloyl CoA. As carnitine conjugation is thought to be the only detoxification metabolic route in canine muscle, under certain circumstances such as carnitine deficiency, the risk of exposure with toxic pivaloyl CoA might increase and the CoASH pool in canine muscle might be exhausted, resulting in toxicity in canine muscle.

The Role of Carnitine Acyltransferases in the Maintenance of Cell Function

Carnitine acyltransferases catalyse equilibria between acyl-CoA esters and the respective acylcarnitines. Therefore, they act not only as pathway enzymes, but also as modulators of acyl-CoA concentrations within individual sub-cellular compartments. Because acyl-CoA esters are potent biologically active metabolites, carnitine acyltransferase activities are potentially able to affect a diverse range of physiological processes, ranging from insulin secretion, to appetite control, and insulin sensitivity of tissues. The distinctive subcellular distributions of the different types of carnitine acyltransferases also enables them to participate in the transfer of acyl moieties across intracellular membranes, and of particular acylcarnitine esters across the plasma membrane and into the plasma. Pharmacological strategies that make use of these properties to improve cell function are discussed.

The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum.

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS-1 cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT1. Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT1 were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT1 into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained an 88 kDa protein (p88) which was recognized by an anti-L-CPT1 antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT1. Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was as abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT1 is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT1 that is derived from mitochondria, possibly from membrane contact sites.

A Novel Brain-Expressed Protein Related to Carnitine Palmitoyltransferase I

Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

Microsomal malonyl-CoA-sensitive carnitine acyltransferase

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.

Microsomal malonyl-CoA-sensitive carnitine acyltransferase.

Liver microsomes contain two carnitine acyltransferase activities. One of these has properties closely corresponding to those of 88 kDa mitochondrial carnitine palmitoyltransferase-1 (CPT-1). Antisera against CPT-1 cross-react with an 88 kDa microsomal protein, suggesting that CPT-1 may be targeted to both microsomal and mitochondrial membranes. However, no experiments using cDNAs corresponding to CPT-1 involving in vitro translation with microsomes or involving in vivo COS-1 cell transfection provided any evidence to support this hypothesis.

Mitogenic effect of retinoid X receptor agonists in rat liver

(E)-2-[2-(5,6,7,8-Tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-l-yl]-4-thiophenecar-boxylic acid (AGN 191701) and other retinoid X receptor (RXR)-selective agonists were observed to cause Hepatomegaly in rats. The purpose of the present study was to understand the biochemical basis of RXR agonist-induced hepatomegaly. Male Fischer rats were implanted s.c. with osmotic pumps containing 5-bromo-2′-deoxyuridine (BrdU) and treated by gavage with 0, 60, or 180 μmol/kg/day of AGN 191701 for 3 days. AGN 191701 caused dose-dependent hepatomegaly in the absence of hepatic necrosis and increased hepatocyte BrdU labeling index (LI). To determine if AGN 191701-induced hepatic hyperplasia was sustained, rats were treated by gavage with 60 μmol/kg of AGN 191701 for up to 7 days and exposed to BrdU via osmotic pump on days 1–3 or on days 6–8. Hepatocyte LI and mitotic index were increased only in rats exposed to BrdU on days 1–3, indicating that AGN 191701-induced hepatocyte proliferation was transient. The receptor specificity of this mitogenic effect was tested by co-treating rats for 2 days with various retinoids and BrdU. 2-(5,6,7,8-Tetrahydro-5,5,8, 8-tetramethyl-2-naphthyl)-2-(4-carboxylphenyl)-l,3-dioxolane (SR11237), an RXR-selective agonist, and (E)-5-[2-(5,6,7,8-tetrahydro-3,5,5,8-pentamethyl-2-naphthyl)propen-l-yl]-2-thiophenecarboxylic acid (AGN 191659), a retinoic acid receptor (RAR)/RXR pan-agonist, both increased hepatocyte LI. Two RAR-selective agonists, all- trans-retinoic acid and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen-l-yl] benzoic acid (TTNPB), did not affect hepatocyte LI. To determine if RXR agonists have biochemical effects in common with a peroxisome proliferator, various endpoints were measured 24 hr after two daily treatments with AGN 191701, SR11237, or clofibrate. While all three compounds induced hepatic acyl CoA oxidase activity, only clofibrate increased hepatic carnitine acyl transferase activity and lowered serum triglycerides. Taken together, these data show that RXR-selective agonists but not RAR-selective agonists cause hepatomegaly accompanied by hepatocyte mitogenesis in rats. The fact that RXR agonists have some biological effects distinct from RAR agonists and clofibrate suggests that RXR-selective agonists may have unique therapeutic applications.

Time Course of the Effects of a High-Fat Diet and Voluntary Exercise on Muscle Enzyme Activity in Long-Evans Rats

CHENG, B., O. KARAMIZRAK, T. D. NOAKES, S. C. DENNIS, AND E. V. LAMBERT. Time course of the effects of a high-fat diet and voluntary exercise on muscle enzyme activity. PHYSIOL BEHAV 61(5) 701–705, 1997.—This study examined the time course of the effects of a high-fat diet and voluntary running exercise on rat skeletal muscle carnitine acyltransferase (CAT), β-hydroxy-acyl-CoA dehydrogenase (HAD), and citrate synthase (CS) activities. Sixty male Long-Evans rats were randomly allocated to receive either a standard (12% fat by energy) laboratory chow diet (CHOW) or a high-fat (76% by energy) diet (HFD) and placed in running wheels for up to 6 weeks. Energy intakes and weekly voluntary running distances were similar in the CHOW and HFD rats. In both groups, weekly training distance more than doubled from week 4 to week 6. However, increased training had little influence on soleus (s) CAT(s), HAD(s), and CS(s) activities. CAT(s) and HAD(s) activities were higher in the HFD rats than in the CHOW rats from 2 weeks onward ( p < 0.005), and CS(s) activities were not different between groups and remained constant over time. In contrast, increased training distance after 4 weeks in the CHOW rats resulted in an increase in deep vastus (v) CAT(v) activities to values similar to those in HFD rats prior to increases in training volume ( p < 0.005) but had no effect on their HAD(v) and CS(v) activities. Increases in HAD(v) and CS(v) activities with increased training volume were only seen in the HFD rats ( p < 0.005). HAD(v) activities and HAD/CS(v) activity ratios correlated with training distance in the HFD rats only ( p < 0.001 and p < 0.01, respectively). These results suggest that a high-fat diet improves the β-oxidation capacity of rat predominantly slow-twitch soleus muscle and enhances the effects of modest levels of training on the mitochondrial density and β-oxidation capacity of rat deep vastus mixed fast- and slow-twitch muscles.

Effect of membrane environment on the activity and inhibitability by malonyl-CoA of the carnitine acyltransferase of hepatic microsomal membranes.

We have investigated the extent to which membrane environment affects the catalytic properties of the malonyl-CoA-sensitive carnitine acyltransferase of liver microsomal membranes. Arrhenius-type plots of activity were linear in the absence and presence of malonyl-CoA (2.5 microM). Sensitivity to malonyl-CoA increased with decreasing assay temperature. Partly purified enzyme displayed an increased K0.5 (substrate concentration supporting half the maximal reaction rate) for myristoyl-CoA and a reduced sensitivity to malonyl-CoA compared with the enzyme in situ in membranes. Reconstitution with liposomes of a range of compositions restored the K0.5 for myristoyl-CoA to values similar to that seen in native membranes. The lipid requirements for restoration of sensitivity to malonyl-CoA were more stringent. When animals were starved for 24 h the specific activity of carnitine acyltransferase in microsomal membrane residues was increased 3.3-fold, whereas sensitivity to malonyl-CoA was decreased to 1/2.8. When enzymes partly purified from fed and starved animals were reconstituted into crude soybean phosphatidylcholine liposomes there was no difference in sensitivity to malonyl-CoA. When partly purified enzyme from fed rats was reconstituted into liposomes prepared from microsomal membrane lipids from fed animals it was 2.2-fold more sensitive to malonyl-CoA than when reconstituted with liposomes prepared from microsomal membrane lipids from starved animals. This suggests that the physiological changes in sensitivity to malonyl-CoA are mediated via changes in membrane lipid composition rather than via modification of the enzyme protein itself. The increased specific actvity of acyltransferase observed on starvation could not be attributed to changes in membrane lipid composition.

The effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition on tissue levels of carnitine and carnitine acyltransferase activity in the rabbit.

Recently, a new class of lipid lowering agents [3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors] was introduced into clinical practice. The use of these agents could lead to a secondary deficiency in carnitine, which may manifest clinically as a myalgia/myositis-a side effect that is occasionally seen with this class of drugs. In the present study, we examined the effect of an HMG-CoA reductase inhibitor (lovastatin) on serum and tissue levels of carnitine and carnitine acyltransferase activities in the rabbit. Rabbits (n = 6) were fed chow containing lovastatin (30 mg/d) for 16 wk. Blood was collected and tissues (liver, heart, and skeletal muscle) harvested at sacrifice. Free and total carnitine were measured in serum and tissues by a radioenzymatic method. Carnitine acetyltransferase and carnitine palmitoyltransferase (CPT) activities were determined and expressed relative to DNA. Serum free (24.0 +/- 2.6 vs. 29.4 +/- 3.1 microM) and total (35.1 +/- 4.7 vs. 52.8 +/- 8.8 microM) carnitine levels increased significantly with 16 wk of treatment. This increase in total carnitine was mainly due to an increase in the levels of serum acylcarnitine (12.7 +/- 3.1 vs 26.5 +/- 5.7 microM). Tissue levels of total carnitine were significantly decreased by the treatment. Carnitine acetyltransferase was unaffected by the treatment, whereas there was a significant increase in the activity of CPT in the liver and heart.

Spontaneous development of fatty liver in ferrets in a toxicology study

Ferrets were maintained for 12 months on different diets (A, meat and biscuit; B, all meat; C, meat and fish; D, high fibre) to ascertain the cause of spontaneous development of fatty liver. High hepatic triglyceride contents resulted on diets B = C > D; whereas ferrets on diet A (control) showed no accumulation of lipid in liver. Serum triglyceride and total cholesterol were unchanged by diet. These ferrets (F 0 generation) were mated with ferrets on the same diet and the offspring (F 1 generation), maintained on the same diets as the parents, were killed at 12 months and the livers studied similarly. Histology showed that hepatic lipid accumulation in the F 1 generation was identical with that in the same dietary groups of the F 0 generation; liver glutathione (GSH) reductase and thiobarbituric acid-reacting substances (an index of lipid peroxidation) were increased in ferrets maintained on diets B, C and D; liver GSH concentration and GSH peroxidase activities were unchanged. Other ferrets fed a high-fat diet (diet A plus 20% w/w beef suet) for 18 days exhibited hepatic lipid accumulation and decreased hepatic cyanide-insensitive palmitoyl CoA oxidation (−30%), but hepatic lauric acid hydroxylation and carnitine acyl transferase activities were unchanged. These data indicate that ferrets on high-fat diets show no increased rates of liver fatty acid oxidation, as seen in rats, but instead accumulate triglyceride in the liver with some degree of lipid peroxidation.

CDNA Cloning and Baculovirus Expression of the Human Liver Endoplasmic Reticulum P58: Characterization as a Protein Disulfide Isomerase Isoform, but Not as a Protease or a Carnitine Acyltransferase

The function of a 58-kDa liver microsomal protein (P58) is controversial. To help clarify the physiological function of this protein, particularly in humans, a full-length human liver cDNA clone was isolated, sequenced, and expressed in milligram quantities with the use of a baculovirus expression system. The deduced amino acid sequence of the mature protein contained two thioredoxin-like active site motifs (CGHC) and in its C-terminus a nuclear localization motif (KPKKKKK), and an ER-retention/retrieval motif (QEDL). The mature form of human P58 shared 95% amino acid sequence identity with the deduced amino acid sequences of a bovine liver cDNA, 93% with a murine B lymphocyte cDNA, and 91% with a rat basophilic leukemia cell cDNA. In contrast to reports on the activities of nonhuman forms of P58, the purified expressed human P58 showed no carnitine acyltransferase or protease activities. However, it did have protein disulfide isomerase activity, indicating that the physiological activity of human liver P58 may be attributed, at least in part, to this activity.

Inhibition of liver microsomal carnitine acyltransferases by sulphonylurea drugs

The sulphonylureas glibenclamide and tolbutamide inhibited carnitine acyltransferase activities in rat liver microsomes. Glibenclamide was a more potent inhibitor than tolbutamide. The effect of tolbutamide on the malonyl-CoA-inhibitable transferase was influenced by the phospholipid/detergent environment whereas the effect of glibenclamide was not. Glibenclamide was a more potent inhibitor of the malonyl-CoA-inhibitable transferase than of the malonyl-CoA-insensitive enzyme. The extent of inhibition of the malonyl-CoA-inhibitable transferase by tolbutamide was similar to its effect on VLDL triacylglycerol secretion as reported by Wiggins and Gibbons [Biochem. J. 284 (1992) 457–462] possibly supporting the suggestion that microsomal carnitine acyltransferases are involved in VLDL triacylglycerol assembly/secretion.

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity. Hofstra, A. H., King, L. M., and Walker, R. M. (1995). Fundam. Appl. Toxicol. 27, 277-286. CI-924 (5,5′-[[1,1′-biphenyl]-2,5-diylbis(oxy)]bis[2,2-dimethylpentanoic acid]), a lipid lowering agent, was previously shown to be hepatotumorigenic in male and female B6C3F1 mice but not in male and female albino Wistar rats. To determine if the difference between the species in tumorigenic response correlated with the extent of peroxisome proliferation or microsomal changes the effects of CI-924 on liver were characterized in rats and mice. CI-924 doses of 0, 5, 25, 75, and 150 mg/kg were administered in the diet for 4 weeks to B6C3F1 mice and albino Wistar rats. Peroxisomal β-oxidation activity was significantly increased in all groups at doses of 25 mg/kg or higher and was induced up to 25 times in male rats. Peroxisomal carnitine acyltransferase and acyl-CoA oxidase activities were also increased, with the greatest induction observed in male rats. Catalase activity quadrupled in rats and doubled in mice. Serum liver enzyme activities were unchanged with the exception of 5′-nucleotidase which was elevated in mice and decreased in male, but not female, rats. Glutathione S-transferase decreased in the males of both species and glutathione peroxidase increased in the mice. Cytochrome P450 4A1 increased in both species at doses of 25 mg/kg or greater and correlated with increased lauric acid hydroxylation. The high degree of peroxisome proliferation in male rats was unexpected in light of the lack of tumorgenicity demonstrated in a previous 2-year study and these results indicate that early peroxisome proliferation alone is not always a good predictor of hepatocarcinogenicity.

Effect of various n − 3/ n − 6 fatty acid ratio contents of high fat diets on rat liver and heart peroxisomal and mitochondrial β-oxidation

The present work extends tissue investigations previously performed in rat gastric mucosa on lipid metabolism alterations caused by n − 3 and n − 6 fatty acid-enriched diets [13–15]. Liver and heart tissues are here studied and demonstrated to undergo, upon exposure to high fat diets with various n − 3/ n − 6 fatty acid ratio contents, biochemical and morphological changes which may be enumerated as follows: (1) Rat liver peroxisomal prostaglandin E 2, fatty acid but not bile acid β-oxidation rates are enhanced, especially upon the diet with the higher n − 3/ n − 6 fatty acid ratio. Mitochondrial β-oxidation rates are little or not affected by the high fat diets. (2) Rat liver carnitine acyltransferases are stimulated by the high fat diets, the more rich the n − 3 fatty acid content, the more pronounced the stimulatory effect. (3) Rat heart peroxisomal and mitochondrial β-oxidation rates were increased in animals receiving the n − 3 fatty acid-enriched diet. At a low n − 3/ n − 6 fatty acid ratio content of the diet, these oxidizing rate values were in control range. The carnitine acyltransferase activities were increased in rat heart to different extents, depending on the n − 3/ n − 6 fatty acid ratio content of the diet. (4) Ultrastructural examination and morphometric determinations on hepatocytes from rats receiving the diets with the lowest and the highest n − 3/n − 6 fatty acid ratio contents disclose that in the latter case the numbers and fractional volumes of peroxisomes and mitochondria are significantly higher than in the former case.

Regulation of the long-chain carnitine acyltransferases.

Long-chain carnitine acyltransferases are a family of enzymes found in mitochondria, peroxisomes, and endoplasmic reticulum that catalyze the exchange of carnitine for coenzyme A in the fatty acyl-CoA. Conversion of the fatty acyl-CoA to fatty acylcarnitine renders the fatty acid more permeable to the various cellular membranes. The mitochondrial carnitine palmitoyltransferases are considered important in the regulation of mitochondrial beta-oxidation of long-chain fatty acids. However, palmitoylcarnitine produced by peroxisomal carnitine octanoyltransferase or by microsomal carnitine palmitoyltransferase is not different from that produced by the mitochondrial enzyme. Therefore, for there to be control of fatty acid oxidation by the long-chain carnitine acyltransferases, there would have to be some mechanism to coordinately regulate these varied enzymes. The first system of regulation involves inhibition by malonyl-CoA, an intermediate in the synthesis of fatty acids. Malonyl-CoA inhibits long-chain carnitine acyltransferase activity by all three enzymes at similar concentrations in the physiological range. In addition, the mitochondrial and peroxisomal enzymes are known to be regulated at the level of mRNA transcription by a number of shared factors. Although the microsomal enzyme is less well studied, there does, indeed, appear to be a pattern of coordinate regulation for this system.