INTRODUCTION

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The peroxisomes and the mitochondria are two separate fatty acid -oxidation systems having distinct roles in fatty acids catabolism, energy production, and substrate cycling within the cell. The -oxidation system of the peroxisomes, unlike that of the mitochondria, is not coupled to oxidative phosphorylation and is an important source of acetyl (2-carbon) units for the synthesis of long chain fatty acids by chain elongation (1). Fatty acid cycling of polyunsaturated fatty acids in the peroxisomes has been shown to play an important role in the metabolism of essential fatty acids (2). The role of fatty acid cycling by chain shortening/elongation of saturated fatty acids is not well known. Because of recycling of label and the lack of proper isotopic methods, the study of chain shortening/elongation of nonessential fatty acids has been difficult.

Recently, we have developed stable isotope methods for the study of essential and nonessential fatty acid metabolism using uniformly labeled compounds and mass spectrometry (3, 4). For example, chain shortening of [U-¹³C]stearate produces palmitate with a mass shift of 16 daltons due to ¹³C carbons, and the elongation of [U-¹³C]stearate produces arachidate (C20:0) and behenate (C22:0) with a characteristic mass shift of 18 daltons. Thus, chain shortening and elongation can be measured by the formation of these unique isotopomer species. We report here a study of chain shortening and elongation of stearate (C18:0) and the role of activation of peroxisome oxidation with troglitazone, a peroxisome proliferator-activated receptor (PPAR)¹ ligand, on stearate metabolism in HepG2 cells in culture using uniformly ¹³C-labeled stearate and oleate and mass isotopomer analysis.

	MATERIALS AND METHODS

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Tissue Culture-- Human hepatoma cell line HepG2 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and it was grown in 75-ml flasks in Dulbecco's modified Eagle's medium augmented with 10% fetal bovine serum (5). When the cells were ~50% confluent (~2.5 × 10⁶ cells/flask), the medium was removed, the cells were washed with phosphate-buffered saline, and the appropriate medium containing U-¹³C-fatty acids was added as described below to begin the experiment. The incubation lasted 72 h with changes of fresh medium daily.

Isotopes and Drugs-- [U-¹³C]Stearic acid and [U-¹³C]oleic acid were obtained from Martek Biosciences (Columbia, MD) as their sodium salts. They were dissolved in warm water and added separately to the culture medium at a concentration of 0.7 mmol/liter. Troglitazone was obtained from Park Davis Pharmaceuticals (Ann Arbor, MI). It was dissolved in dimethyl sulfoxide (Me₂SO) and added to the appropriate flasks to a final concentration of 50 µM. The same volume of Me₂SO was added to the flasks that did not contain troglitazone. Each incubation condition was performed in triplicate, and each analysis was also run in triplicate. During the experiment, HepG2 cells doubled to approximately 0.5 to 1 × 10⁷ cells per plate, and remained 80-90% viable after harvest.

Extraction of Lipids from the Cell Pellet-- Fatty acids were extracted according to the method described by Lowenstein et al. (6). The cell pellet was saponified with 1 ml of 30% KOH:ethanol (v:v, 1:1) at 70 °C overnight. Neutral lipids were first removed with petroleum ether extraction. The solution containing the saponified fatty acids was then acidified, and palmitate and other fatty acids were recovered with another petroleum ether extraction. Fatty acids were methylated with 0.5 N HCl in methanol (Supelco, Bellfonte, PA) for GC/MS analysis (7).

GC/MS Analyses-- Fatty acids were analyzed as their methyl esters. Palmitate, palmitoleate, stearate, and oleate were separated on HP5840A GC with a 3-foot SP2330 glass column using temperature programming. The GC conditions were as follows. Helium flow rate was 20 ml/min and the initial temperature was held at 180 °C for 1 min, and then the oven temperature was programmed to increase at 3 °C/min to a final temperature of 210 °C. Under these GC conditions, the retention times for palmitate, palmitoleate, stearate, and oleate were 3.1, 3.7, 5.1, and 5.7 min, respectively. The temperature of the GC to mass spectrometer interface was maintained at 275 °C and the source temperature at 200 °C. Mass spectra were obtained on the HP5985 mass spectrometer using electron ionization at (70 eV) and selected ion monitoring. Ion clusters monitored for the quantitation of isotopomers of palmitate were m/z 269-276 and 286-290 with m + 0 at m/z 270 and m + 16 at m/z 286. The corresponding clusters monitored for palmitoleate were m/z 236-240 and 251-255; stearate, m/z 298-302 (m + 0 at m/z 298) and m/z 312-316 (m + 18 at m/z 316); and oleate, m/z 264-267 and 282-285. Normalized spectra of "unlabeled"² and [U-¹³C]stearate and [U-¹³C]oleate are shown in Fig. 1.

Data Analysis-- Mass spectra were acquired in the traditional "normalized"³ format. For the purpose of determining enrichment and fractional uptake and conversion, the mass spectra were expressed as molar fractions, i.e. the fraction of molecules with a particular mass. This is achieved by dividing the intensity of each peak by the sum of intensities of all relevant peaks of that compound. The expression of spectral data as molar fraction allows the determination of average mass (or average molecular weight) by the sum of the products of molar fraction and mass over the relevant mass range (8).⁴ Since the chance of [¹³C]acetyl-CoA condensing to form uniformly labeled fatty acids is almost zero, the sum of individual fractions of ions in the clusters corresponding to the uniformly labeled fatty acids gives the fraction of molecules converted by chain elongation or shortening.⁵

Determination of Precursor Enrichment and de Novo Synthesis-- Finally, the enrichment of the acetyl-CoA pool from -oxidation of uniformly labeled fatty acids can be determined from the distribution of mass isotopomers of palmitate. De novo synthesis of palmitate produces palmitate with 2, 4, or 6 ¹³C atoms (m₂, m₄, and m₆). The distribution of these mass isotopomers has previously been shown to conform to that of a binomial distribution (10, 11). Thus, the acetyl-CoA enrichment can be obtained from the consecutive mass isotopomer ratio m₄/m₂ according to the formula: m₄/m₂ = (n  1)/2 p/q or 3.5 p/q, where n is 8, the number of acetyl units in palmitate, p is the enrichment of [1,2-¹³C]acetyl-CoA, and q the unenriched acetyl-CoA. Once the precursor enrichment is determined, fractional synthesis can be calculated by dividing the observed to the predicted mass isotopomer (m₂) fraction (10).

	RESULTS

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Enrichment of Labeled Precursors-- The spectra of uniformly labeled and "unlabeled" stearate and oleate are shown in Fig. 1. The molecular ion for saturated fatty acid is the methyl ester itself. The distribution of m + 1, m + 2, ... isotopomers follows that of a binomial distribution reflecting the natural abundance of ¹³C. The spectrum of oleate does not follow that of a simple binomial distribution. The spectrum of oleate is more complex than that of stearate showing the loss of -OCH₃ and -HOCH₃⁶ from the methyl group. The molecular ions of stearate and oleate were shifted by 18 daltons in the uniformly labeled fatty acids. The presence of ¹²C resulted in the formation of m + 17 peaks in the spectra of [U-¹³C]stearate and oleate. The ¹³C enrichment of these labeled compounds can be determined as the change in average molecular weight using the average mass approach (8). We found the enrichment of [U-¹³C]stearate and [U-¹³C]oleate to be 97.6 and 91.0%, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Normalized mass spectra of methyl esters of uniformly labeled and unlabeled stearate and oleate. The base peaks for unlabeled stearate and oleate were m/z 298 and m/z 264. Uniformly labeled fatty acids all showed the characteristic 18-dalton mass shift (m + 18 peaks).

Pathway Interpretation-- The interconversion of stearate and oleate with palmitate and palmitoleate is outlined in Fig. 2. The horizontal pathways represent chain elongation or shortening and the vertical pathways, desaturation or reduction. When HepG2 cells were provided with [U-¹³C]stearate, chain shortening resulted in the formation of [U-¹³C]palmitate containing 16 ¹³C carbons. Desaturation at the 9 position of [U-¹³C]palmitate and [U-¹³C]stearate gave rise to [U-¹³C]palmitoleate and [U-¹³C]oleate (Fig. 3). The conversion of oleate to palmitoleate has not been reported. Chain shortening of [U-¹³C]oleate resulted in the formation of a 7 C16:1 fatty acid, which has to be further processed to give the 9 fatty acid ([U-¹³C]palmitoleate). Reduction of the carbon-carbon double bond of [U-¹³C]oleate produced a small amount of [U-¹³C]palmitate and [U-¹³C]stearate (Fig. 4). The operation of these pathways of fatty acid interconversion in HepG2 cells in culture was clearly demonstrated by the presence of m + 16 and m + 18 isotopomers of these fatty acids as shown.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Metabolic pathways leading to the conversion of C18 fatty acids to C16, C20, and C22 fatty acids. The horizontal arrows represent pathways of peroxisomal -oxidation and chain elongation. The vertical arrows represent pathways of stearoyl desaturase and the reduction of the 9 double bonds. The conversion of oleate to palmitoleate is represented by a broken arrow indicating a multistep process. Entry of the uniformly labeled fatty acids are indicated by the open arrows.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Mass spectra of methyl esters of palmitate, palmitoleate, and oleate isolated from HepG2 cells incubated with [U-13C]stearate. The base peaks for palmitate, palmitoleate, and oleate were m/z 270, 236, and 264, respectively. A substantial percent of palmitate was produced by chain shortening of [U-13C]stearate. This is shown by the presence of [U-13C]palmitate (m + 16). Subsequent desaturation of [U-13C]palmitate produced m + 16 palmitoleate. The bottom panel shows the action of stearoyl desaturase forming m + 18 oleate.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Mass spectra of methyl esters of palmitate, palmitoleate, and stearate isolated from HepG2 cells incubated with [U-13C]oleate. The base peaks for palmitate, palmitoleate, and stearate were m/z 270, 236, and 298, respectively. There was little formation of palmitate from oleate, which requires chain shortening and reduction. A substantial percent of palmitoleate molecules with mass shift of 16 daltons (m + 16) was produced by chain shortening of [U-13C]oleate. The lack of reductase action was also seen in the conversion of oleate to stearate (bottom panel).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Mass spectra of methyl esters of arachidate (C20:0) and behenate (C22:0) isolated from HepG2 cells incubated with [U-13C]stearate. The base peaks for arachidate and behenate were m/z 326 and 354. The ability to convert [U-13C]stearate to arachidate and behenate in HepG2 cells was demonstrated by the presence of these fatty acids with the corresponding m + 18 mass shift (m/z 344 and 372). Fatty acids with m + 20 and m + 22 were also found in addition to those of m + 18, indicating a high degree of recycling of labeled acetyl-CoA generated from -oxidation. The acetyl-CoA enrichment of the precursor pool for chain elongation can be estimated by comparing the m + 20 to m + 18 isotopomers of arachidate to be 35%. In other words, 35% of the acetyl-CoA was derived from -oxidation of [U-13C]stearate.

Effects of Troglitazone on Fatty Acid Metabolism-- The contributions of uptake of labeled fatty acids and their conversion by chain shortening/elongation to the fatty acids of HepG2 cells are shown in Fig. 6. When HepG2 cells were supplied with 700 µM of stearate or oleate, the uptake of medium fatty acids accounted for 93 and 84% of the cellular stearate and oleate suggesting suppression of de novo synthesis of these fatty acids. The incorporation of medium fatty acids was not affected by troglitazone (Fig. 6A). Under the same incubation medium, 16% of the palmitate (C16:0) was derived from [U-¹³C]stearate and 26% of C16:1 from [U-¹³C]oleate by chain shortening. Such contributions were significantly increased by troglitazone to 23.6 and 36.6%, respectively (p < 0.001) (Fig. 6B). Chain elongation was much more active than chain shortening when cells were supplied with additional stearate. Chain elongation accounted for 88.3% of arachidate and 63.7% of behenate molecules. These fractions were reduced in the presence of troglitazone to 84.0% and 54.2% respectively (p < 0.001).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of troglitazone on the contribution of A, uptake; B, chain shortening; and C, chain elongation of uniformly labeled stearate and oleate to saturated and monounsaturated fatty acids. Fatty acids from untreated cells are shown by the open bars and those from troglitazone-treated cells by the filled bars. Uptake and conversion of labeled fatty acid are expressed as percent of the respective fatty acid molecules. Values are the mean ± S.D. of three experiments. The asterisk (*) denotes significant differences (p < 0.001) between troglitazone-treated versus untreated cells. Panel A shows the uptake of uniformly labeled stearate and oleate, which accounted for >80% of those fatty acids in the cells. Panel B shows the contribution of chain shortening of stearate and oleate to palmitate and palmitoleate. Troglitazone significantly increased the contribution of chain shortening to these fatty acids. Panel C shows that [U-13C]stearate was preferentially used for chain elongation in the synthesis of arachidate and behenate. Troglitazone significantly inhibited (p < 0.001) the chain elongation of [U-13C]stearate.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Percent contribution of stearoyl desaturase and reductase to the production of saturated and monounsaturated fatty acids. About 70% of oleate was produced from the desaturation of [U-13C]stearate. Whereas, very little monounsaturated fatty acids were reduced to produce palmitate or stearate. These pathways were not affected by troglitazone treatment.

	DISCUSSION

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Supplementation of diet with fatty acids is known to regulate endogenous synthesis of lipids. Transcriptional regulation of lipogenic enzymes, hepatic fatty acid synthase, malic enzyme, and glucose-6-phosphate dehydrogenase, in hepatocytes by fatty acids has been extensively studied (12-14). The effects on enzymes of de novo lipogenesis appeared to be specific for polyunsaturated fatty acids of the n-6 and n-3 families, and saturated and monunsaturated fatty acids did not have the same inhibitory effects on these lipogenic enzymes (15). Fatty acids can also be recycled by chain shortening/elongation of other fatty acids. The existence of such a fatty acid cycling system has been well documented for essential fatty acid metabolism. Fatty acid interconversion requires the participation of peroxisomal, microsomal and endoplasmic reticulum systems involving enzymes of -oxidation, desaturases, isomerases and reductases. The function of some of these enzymes are often inferred from their precursor-product relationship, and the substrate specificity of these enzymes have not been well characterized (2). We demonstrated here that such a system also exists for the non-essential fatty acids. However, the enzymes involved in the reactions of the cycling of nonessential fatty acids remain to be elucidated. The present study examined the impact of stearate and oleate supplementation on fatty acid cycling and de novo synthesis of nonessential fatty acids. With supplementation of 0.7 mM of stearate or oleate, we observed a number of very specific effects on the synthesis of a series of C16, C18, C20, and C22 fatty acids. When stearate and oleate were provided in relative excess, the uptake of these fatty acids accounted for over 80% of these fatty acids found in HepG2 cells. The conversion of these fatty acids by chain shortening and desaturation were also important in the production of palmitate and palmitoleate and oleate. By inference, de novo lipogenesis of these fatty acids was suppressed. De novo lipogenesis of palmitate in HepG2 cells was previously shown to be 80% for the same period of incubation (5). When supplied with stearate and oleate, de novo lipogenesis was suppressed to about 40%.

Troglitazone is a thiazolidinedione compound which is known to bind with the PPAR. The binding of troglitazone to PPAR stimulates peroxisome proliferation, and induces the expression of a number of genes regulated by the peroxisome proliferator response elements (16, 17). Among these genes are the acyl-CoA oxidase and dihydroxyacetone phosphate acyl transferase, which are enzymes of lipid oxidation and biosynthesis (18, 19). The effect of troglitazone on lipid metabolism was previously studied in hepatocytes isolated from troglitazone treated rats (20, 21). Mitochondrial -oxidation as measured by the release of radioactive CO₂ or the release of acid soluble product (ketone bodies) from ¹⁴C-labeled palmitate or oleate was reduced by troglitazone treatment. However, conflicting observations were reported by Shimabukuro et al. (22) showing an increase in mitochondrial -oxidation of [³H]palmitate in islets of troglitazone treated Zucker diabetic fatty rats. The discrepancies in these observations may be attributed to the difference in the isotopic methods used or in tissue specific responses. Troglitazone was shown to noncompetitively inhibit mitochondrial and microsomal acyl-CoA synthase of rat hepatocytes (20) and decrease the mRNA of glycerol-3-phosphate acyltransferase and acyl-CoA synthase mRNA content in islets of Zucker diabetic fatty rats (21). Subsequently, the esterification of fatty acid to triglycerides and the triglyceride content in cells were inhibited by troglitazone treatment. In the present study, we showed that troglitazone increased chain shortening of C18 fatty acids thus peroxisomal -oxidation of these fatty acids in HepG2 cells in culture. The increased -oxidation resulted in higher ¹³C enrichment of the acetyl precursor pools both for de novo lipogenesis and chain elongation. However, de novo lipogenesis of palmitate as well as the synthesis of arachidate and behenate by chain elongation were significantly inhibited by troglitazone treatment. The action of stearoyl-CoA desaturase activity contributed significantly to the formation of mono-unsaturated fatty acids from palmitate and stearate. The desaturation of saturated fatty acids to mono-unsaturated fatty acids was not affected by troglitazone.

Mitochondria and peroxisomes are the main cellular systems for fatty acids -oxidation. Mitochondrial -oxidation which is coupled to oxidative phosphorylation results in the complete breakdown of the fatty acid to acetyl-CoA, CO₂, high energy phosphate bonds and reducing equivalents. Peroxisomal -oxidation system on the other hand is not coupled to oxidative phosphorylation, and produces acetyl-CoA and the fatty acids of shorter chain lengths. The action of peroxisomal -oxidation system is responsible for chain shortening/elongation of polyunsaturated fatty acids, and for the recycling of acetyl (2-carbon) units and essential fatty acids in the homeostasis of these essential fatty acids (2). The substantial amount of palmitate and palmitoleate formed by chain shortening in our experiments suggests that the peroxisomal system also plays a significant role in the -oxidation of long chain saturated and mono-unsaturated fatty acids, just as in the case of the very long chain fatty acid (23). This process was stimulated by the peroxisome proliferator troglitazone.

Chain shortening/elongation by peroxisomal -oxidation characteristically allows energy to be stored or used without significant changes in the number of fatty acid molecules. Furthermore, chain shortening/elongation is a less "futile" process involving the recycling of 1-3 acetyl units as compared with eight via mitochondrial -oxidation and de novo synthesis of palmitate. Peroxisome -oxidation is potentially an energy saving system for the interconversion of fatty acids needed for membrane lipid synthesis. It is conceivable that these effects of troglitazone on oxidation, interconversion, and synthesis saturated fatty acids play a major role in cellular energy metabolism, and membrane lipid composition and turnover. However, the mechanism relating these effects to its "insulin-sensitizing" effect in the treatment of non-insulin dependent diabetes is not known and deserves further investigation.