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UPRES Lipides et Nutrition EA2422, Faculté des Sciences Gabriel, Université de Bourgogne, 21000 Dijon, France; and * INRA, Unité de Nutrition Lipidique, 21034 Dijon Cedex, France
2To whom correspondence should be addressed. E-mail: pclouet{at}u-bourgogne.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • acetyl-CoA carboxylase • acyl-CoA oxidase • carnitine palmitoyltransferase I • malonyl-CoA • regulation
Conjugated linoleic acids (CLA)3 are a group of dienoic derivatives of linoleic acid that are mainly produced in ruminants (1,2). These derivatives have exhibited a variety of unique properties such as anti-cancer (2–4), anti-atherogenic (5), and immune response enhancing effects (6) in animal models. CLA have also reduced total body fat content in mice (7,8), rats, and chickens (9). In natural products, CLA are essentially represented by cis-9,trans-11-C18:2 (2,10), abbreviated CLA1, while the trans-10,cis-12 homologue (CLA2) is mainly found in synthetic products utilized in most feeding studies (11–13). In CLA-fed mice, both forms were chiefly recovered in triacylglycerols (TAG) and, to a far less extent, in phospholipids (PL) of rat hepatocytes (14). CLA1 was demonstrated to be one of the most avid ligands yet described for PPAR
, with CLA2 being less potent (12). However, in contrast to clofibrate, these CLA did not act as classical peroxisome proliferators in rats (12). In mice it has been shown that a diet enriched with a mixture of CLA (7,8,15) or CLA2 alone, but not CLA1 (16), caused the reduction in body fat mass and a marked liver steatosis. This latter observation was rather surprising because CLA2 has been shown to be oxidized by normal rat liver mitochondria more than CLA1 (17) and was therefore likely to lose its properties more rapidly. Moreover, it was recently shown that the steatosis did not result from the impairment of liver lipoprotein secretion (18). These facts prompted us to investigate to what extent dietary CLA1 and CLA2 were recovered in lipids of liver and periepididymal adipose tissues (PAT) and to determine whether biochemical alterations and enzymatic inductions related to key steps of fatty acid (FA) oxidation were involved in the development of hepatic steatosis in CLA2-fed mice. We studied food-deprived mice in which FA oxidation rates are greater and much less affected by lipogenic activities, through malonyl-CoA synthesis, than in mice that had recently eaten.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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cis-9,trans-11-Linoleic-, trans-10,cis-12-linoleic-, and cis-9-oleic acids were esterified as TAG by Natural Lipids Ltd. AS and mixed with a basal diet to provide CLA1, CLA2, or control diet, respectively (Table 1). Carnitine was a gift from Dr. G. Lavianne (Sigma-Tau). [1-14C]Palmitic acid as well as [14C]acetyl-CoA was purchased from Perkin–Elmer Life Sciences. L-[Methyl-14C]carnitine and NaH[14C]CO3 were supplied by Amersham Biosciences. Hyamine and Ultima Gold XR were specific products provided by Perkin–Elmer. Biochemicals were from Sigma-Aldrich.
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French guidelines for the use and care of laboratory animals were followed. C57BL/6J male mice (7 wk old), supplied by Harlan, were housed in pools of 4 in plastic cages. They were fed the control diet for 1 wk and were then allocated for 4 wk to the control, CLA1, or CLA2 diet. These diets were given as fresh pellets 3 times a week. At the end of the 4-wk experiment, mice were deprived of food for 16 h and then anesthetized with isoflurane. Blood samples were collected from the abdominal vena cava. Pieces of liver tissues were used immediately or quickly frozen in liquid nitrogen and stored at -80°C. All PAT were dissected by the same operator.
Isolation of mitochondrial fractions
Large pieces of liver were cut finely in ice-cold 0.25 mol/L sucrose medium containing 1 mmol/L EGTA and 10 mmol/L Tris/HCl, pH 7.4, rinsed 5 times in the same medium, blotted with absorbent paper, and weighed. Tissues were diluted (1:80, w:v) and homogenized in the chilled sucrose medium by only 4 strokes of a Teflon pestle rotating at 300 rpm in a cooled Potter-Elvehjem homogenizer (Kontes). The homogenate was centrifuged at 2000 x g for 4 min and the supernatant was immediately centrifuged at 13,000 x g for 3 min. The pellet was resuspended with the homogenization mixture and centrifuged under the preceding conditions. The procedure was repeated once and the resuspended pellet was used as the mitochondrial fraction.
Liver, PAT, and serum lipids
Total lipids of liver and PAT were extracted according to the procedure of Folch et al. (19) and their weights were estimated by gravimetry. In total lipids, the percentages of lipid classes separated on glass rods coated with silica gel were determined by TLC flame ionization detection (20). In order to measure CLA contents, lipid classes were separated from total lipids by classic TLC and recovered by scraping. Their constitutive FA were methylated and quantified by GLC (21). Serum TAG and free fatty acid (FFA) concentrations were estimated using commercially available colorimetric kits, Nos. 337-B (Sigma-Aldrich) and 1383–175 (Roche Diagnostics GmbH), respectively.
Hepatic total carnitine
Part of the frozen liver tissues was powdered in a stainless-steel mortar with liquid nitrogen and treated with 0.1 mol/L KOH at 56°C for 1 h to hydrolyze all carnitine derivatives. After treatment with 100 g/L HClO4 and centrifugation, the neutralized supernatants were used for total carnitine determination by the radiochemical procedure of McGarry and Foster (22).
Mitochondrial and peroxisomal enzyme assays
The presence of mitochondria was assessed by the activities of monoamine oxidase (EC 1.4.3.4) (23) and citrate synthase (EC 4.1.3.7) (24), whereas that of peroxisomes was determined by CN--insensitive palmitoyl-CoA-dependent NAD+ reduction (25), which was described as the peroxisomal fatty acid oxidizing system (PFAOS). Measurements of carnitine palmitoyltransferase I (CPT I) activity were performed at 30°C in a medium containing 25 mmol/L Hepes, pH 7.4, 150 mmol/L sucrose, 60 mmol/L KCl, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 3.9 g/L of fatty acid-poor bovine serum albumin (BSA), and 75 µmol/L palmitoyl-CoA. Mitochondria (0.25 g protein/L medium) were preincubated for 2 min before 0.4 mmol/L L-[3H]carnitine (92.5 GBq/mol) was added. Because the activity of CPT I, contrary to that of CPT II, is almost totally suppressed by malonyl-CoA, actual CPT I activities were calculated by correcting raw values from those obtained with assays containing 150 µmol/L malonyl-CoA. The sensitivity of CPT I to malonyl-CoA inhibition was studied in the presence of 45 µmol/L palmitoyl-CoA and malonyl-CoA at the indicated concentrations. After 4 min, the reaction was stopped by the addition of 1 mL 1.2 mol/L HCl and the acyl-[3H]carnitine produced was extracted with butan-1-ol (26). The associated radioactivity was counted after mixing in Ultima Gold-XR.
Palmitate oxidation
Liver homogenates. The tissues were homogenized in 20 vol of chilled 0.25 mol/L sucrose containing 2 mmol/L EGTA and 10 mmol/L Tris/HCl, pH 7.4, by 4 strokes of a Teflon pestle rotating at 300 rpm in a cooled Potter-Elvehjem homogenizer. Palmitate oxidation rates were measured using 2 media as described in (27), the first allowing mitochondrial and peroxisomal activities to occur and the second allowing peroxisomal activity only. After 2 min of preincubation, the reactions were started by the addition of 120 µmol/L potassium [1-14C]palmitate (55.5 GBq/mol) bound to BSA in a 5:1 mol/L ratio (1.2 mL final volume); they were stopped after 30 min by the addition of 3 mL of 167 g/L HClO4. A conical polyethylene tube containing 0.4 mL of Hyamine was inserted in the incubation vial, before closure, to trap the released 14CO2. After 1 h at room temperature, the radioactivity of Hyamine mixed with Ultima Gold XR was counted. The acidified assays were filtered on Millipore filters (45 µm diameter pores) under very low depression to remove the insoluble unused palmitic acid. The radioactivity of the clarified medium measured after mixing with Ultima Gold XR corresponded to amounts of small molecules (acid-soluble products, ASP) exhibiting the labeling of the carboxylic end of palmitate.
Isolated mitochondria. The incubation medium consisted of 20 mmol/L KH2PO4, pH 7.4, 50 mmol/L KCl, 4 mmol/L MgCl2, 1 mmol/L ATP, 50 µmol/L CoA, 2 mmol/L L-malate, 0.5 mmol/L L-carnitine, and 50 µmol/L potassium [1-14C]palmitate (130 GBq/mol) bound to BSA in a 1.5:1 mol/L ratio. The reaction was initiated with 0.3 mg of mitochondrial protein in 1 mL of medium maintained at 30°C. After 8 min, the reaction was stopped by the addition of 3 mL of 167 g/L HClO4. The radioactivity of CO2 and ASP was measured as for the palmitate oxidation by liver homogenates.
Acetyl-CoA carboxylase (ACC) activity
Mouse livers were cut into small pieces in a chilled medium containing 30 mmol/L sucrose, 220 mmol/L mannitol, 2 mmol/L Hepes, pH 7.5, 0.1 mmol/L EDTA, and 50 mmol/L FNa; they were rinsed 4 times, blotted on absorbent paper, weighed, and homogenized with an Ultra-Turrax homogenizer in the preceding medium (1:5, w:v). The supernatant was treated as in (28) to obtain its maximum ACC activity and assayed for malonyl-CoA synthesis from acetyl-CoA and NaH[14C]CO3 (2.3 TBq/mol) using the same procedure.
Hepatic CoA derivatives
A 200-mg sample of frozen liver was homogenized with 1.5 mL of a chilled 142 g/L HClO4/2 mmol/L dithiothreitol mixture and 20 µL of the clarified perchloric acid extract was immediately injected into a (C18) reverse-phase column (Merck Lichrocart, Superspher; 4 x 250 mm) as in (29). Absorbance measurements of eluted products were made at 254 nm and liver contents in malonyl-CoA, acetyl-CoA, and CoA were assessed by reference to standard solutions.
Hepatic proteins
Protein concentrations of mitochondrial and cytosolic fractions were estimated by the bicinchoninic acid procedure (30) using BSA as a standard.
RT-PCR
Total mRNA was extracted from liver by the Tri-reagent method adapted from the procedure of Chomczynski and Sacchi (31). Tri-reagent was provided by Euromedex. Gene expression was evaluated by semiquantitative RT-PCR from 1 µg of total mRNA using the Access RT-PCR System from Promega. Reverse transcription and cDNA fragment amplification was performed in a Hybaid thermocycler (Omnigene Bioproducts, Inc.). The sequences of the sense and antisense primers designed using Primers! software and synthesized by MWG-Biotech AG were as follows: 5'-GGATCTACAATTCCCCTCTGC-3' and 5'- GCAAAATAGGTCTGCCGACA-3' for L-CPT I, 5'- AGGTATGGCCACTTTGGGA-3' and 5'- AGCTTCAGGGTTTGTCGGA-3' for M-CPT I, 5'- ACTCATCCGCTTTGTTCCTTC-3' and 5'- CTGGGTTTGG-GTATACGAGTTG-3' for CPT II, 5'- GGTGGTATGGTGTCGTACTTGA-3' and 5'- GAATCTTGGGGAGTTTATCTGC-3' for acyl-CoA oxidase (ACO), 5'-AATCGTGCGTGACATCAAAG-3' and 5'-GAAAAGAGCCTCAGGGCAT-3' for ß-actin. The number of amplification cycles was determined according to a kinetic profile. Control tubes containing no RNA template were used to check for contamination. RT-PCR products were resolved in a 17 g/L agarose gel with ethidium bromide and band intensities were measured by densitometric analysis with a Gel Doc 2000 ultraviolet gel documentation system equipped with Quantity One software (Bio-Rad S.A.). For each gene studied, ß-actin was concomitantly amplified and used for normalization.
Statistics
Results are expressed as means ± SEM. When appropriate, data were subjected to one-way ANOVA followed by Student’s t test. When variances and numbers of mice were unequal, means were tested by a Kruskal-Wallis nonparametric test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Preservation of CLA in liver lipids
In the present experiment, in which dietary CLA2 and CLA1 were ingested at equivalent amounts for 4 wk, CLA2 was recovered in significantly lower proportions of total FA (about 90% less) compared to CLA1 in the main lipid classes of liver and PAT. Indeed, CLA2 has been shown to be oxidized to a slightly greater extent than CLA1 by isolated liver mitochondria (17), but the difference in oxidation rates between the isomers could not account for the almost total disappearance of CLA2 from liver lipids. The fact that, compared to CLA1, the CLA2 isomer was proportionally recovered as longer and more desaturated derivatives indicated that CLA2 was a far better substrate for desaturation/elongation reactions (10,33) and for eicosanoid production (34,35) and that one or several of the compounds synthesized may actually exert the properties originally attributed to CLA2.
Gene expression and activities of CPT I and ACO
CLA isomers differ in their effects on gene expression. Some studies suggested that FA and acyl derivatives upregulate the expression of both L-CPT I and ACO genes through PPAR (36–38), but other findings showed that long-chain FA regulate L-CPT I through a PPAR-independent pathway (39,40). Both CLA studied, and CLA1 to a greater extent, were demonstrated to be potent ligands for PPAR (12). In our study, both dietary CLA effectively increased the peroxisomal ACO expression, but CLA1, which was recovered in far greater amounts in liver lipids than CLA2 (Table 2), did not increase L-CPT I gene expression. Therefore, under our experimental conditions, L-CPT I regulation was independent of PPAR activation, as already discussed (39). In mice fed CLA2, which was mainly recovered as desaturated and elongated metabolites [see also review in (41)], the hepatic effects may originate from complex mechanisms, causing at least the induction of L-CPT I and ACO gene expression. Interestingly, transcription of M-CPT I, CPT II, and ACO genes has been shown to be activated by FA via PPAR (42,43) and also significantly via either CLA isomer (this study), which suggests that some effects of both CLA may depend on PPAR (12).
Greater sensitivity of CPT I activity to malonyl-CoA in the livers of CLA1 and CLA2 groups (Fig. 1) may be related to the induction of M-CPT I (Fig. 2), which is far more sensitive to malonyl-CoA compared to L-CPT I (44,45). M-CPT I is normally virtually absent from liver (45), but has been reported to be induced in this organ by the PPAR ligand fenofibrate (46). As previously observed in liver mitochondria from rats (47,48), CPT I activity was shown to be more sensitive to malonyl-CoA inhibition in liver mitochondria from fed than from food-deprived control mice (unpublished data). Interestingly, although treatment with either CLA resulted in greater sensitivity to malonyl-CoA in food-deprived mice, presumably due to induction of M-CPT I expression (see above), this effect was not amplified in fed mice (unpublished data). It will be interesting to determine whether the stimulating effects of both CLA on M-CPT I gene expression are as pronounced in fed vs. food-deprived mice.
Etiology of CLA2-induced liver steatosis
Food intake. Fat accumulation in liver may result from excess energy intake, as observed in high fat–fed mice (49). However, mice fed both CLA isomers gained less weight than controls, despite higher food intake (Table 2), indicative of lower feed efficiency (gram gain/gram food consumed). Thus, excess food intake does not explain the hepatic steatosis in CLA2-treated mice.
Hepatic ß-oxidation. The steatosis of liver in CLA2-fed mice cannot be explained by decreases in mitochondria number and in mitochondrial and peroxisomal FA oxidation capacities. Indeed, CPT I specific activities and FA oxidation capacity rates were greater in both isolated mitochondria and homogenates of liver. Furthermore, in the same group, peroxisomal FA oxidation activities were markedly greater than in controls. Because these activities were expressed per milligram of mitochondrial protein or per gram of liver (see Tables 4, and 6), higher FA oxidation rates should be expected from the whole liver in the CLA2 group, which was heavier. Despite elevated in vitro FA oxidation capacity in the livers of CLA2-treated mice, it is possible that rates of FA combustion in situ were depressed in this group due to an elevated concentration of malonyl-CoA, a potent inhibitor of CPT I which is generated by ACC activity (50). This hypothesis is supported by the fact that liver ACC activity and malonyl-CoA levels per gram of liver were increased in the CLA2 group (Table 5). Notably, ACC activity is increased by insulin (51), and the latter has been reported to be markedly increased by CLA2 in mice (16). However, the assertion that CLA2-induced steatosis may result in part from increased inhibition of CPT I by malonyl-CoA must be reconciled with the fact that malonyl-CoA contents per gram of liver were also elevated in the CLA1-treated mice (Table 6), which did not develop steatosis. It is possible that in CLA2-treated mice in vivo malonyl-CoA inhibition is greater than in CLA1-treated mice. For instance, CLA2-related hyperinsulinemia (16) may increase ACC activity and/or CPT I sensitivity to malonyl-CoA (52,53). Furthermore, the actual cell partitioning of lipogenic vs. inhibitory pools of malonyl-CoA, which likely depend on activities of cytosolic and mitochondrial isoforms of ACC, respectively (54), is not known for control and either CLA1- or CLA2-fed mice. Finally, the cytosolic malonyl-CoA content per gram of wet weight liver may not correspond to the actual pool of malonyl-CoA that specifically inhibits CPT I activity (55). Further research will be required to fully address these issues.
Lipogenesis and TG uptake. Because the fat accumulation within liver cells of CLA2-fed mice appeared not to directly depend on any defect in FA oxidation capacity, the onset of the steatosis might be due to other causes such as (1) a decrease in VLDL secretion, (2) an increase in LDL uptake, and (3) a stimulation of lipogenic activities associated, through malonyl-CoA, with the inhibition of CPT I activity. To address the first two possibilities, it was recently reported that in CLA2-fed mice the VLDL secretion rate was increased, whereas the plasma TAG concentration was decreased, and the levels of LDL-receptor mRNA were clearly greater than in control and CLA1-fed mice (18). These latter studies suggest that TAG-rich lipoproteins were taken up in the CLA2 group to such an extent that the VLDL secretion rate, even when increased, was insufficient to meet the entering lipoprotein flux, which would account for part of the fat deposit within liver cells. On the other hand, the increased lipogenic activities in the same group were also likely to supply liver cells with newly synthesized FA (see ACC activities in Table 5). Indeed, this pathway should be activated by the high levels of blood insulin reported in CLA2-fed mice (16), despite the development of insulin resistance (16,32). In the same group, the long-lasting inhibitory effect of malonyl-CoA on CPT I activity may decrease the actual mitochondrial FA oxidation within liver cells. In that way, even if the total FA oxidation capacity appeared to be greater in vitro, strong control of CPT I activity could decrease the actual in vivo FA oxidation and the nonoxidized FA would account for part of the fat deposit.
Collectively, the data underline the complex roles of both CLA isomers used because these entered catabolic and anabolic pathways to different extents. CLA2 and/or its derivatives increased liver mRNA levels of several mitochondrial and peroxisomal FA oxidation-related enzymes in mice, similar to CLA1. However, induction of L-CPT I expression was specific to CLA2 treatment. Notably, both CLA isomers stimulated liver expression of M-CPT I, normally absent from this tissue. The increased liver mitochondrial and peroxisomal FA oxidation capacities measured in vitro in CLA2-treated mice indicate that the hepatic steatosis in this group is not explained by a lack of FA oxidation per se. Rather, we propose that greater malonyl-CoA generation, and thus increased CPT I inhibition, reduces FA oxidation in vivo. If true, this phenomenon in concert with increased lipoprotein uptake and lipogenesis would largely explain the hepatic steatosis of CLA2-fed mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ACC, acetyl-CoA carboxylase; ACO, acyl-CoA oxidase; ASP, acid-soluble product; BSA, bovine serum albumin; CLA, conjugated linoleic acid; CLA1, cis-9,trans-11-C18:2; CLA2, trans-10,cis-12-C18:2; L/M-CPT I, liver/muscle carnitine palmitoyltransferase I; FA, fatty acid; FFA, free fatty acid; PAT, periepididymal adipose tissue; PFAOS, peroxisomal fatty acid oxidizing system; PL, phospholipid; PPAR, peroxisome proliferator-activated receptor ; TAG, triacylglycerol.
Manuscript received 10 July 2003. Initial review completed 31 July 2003. Revision accepted 19 January 2004.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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