From the Harbor-UCLA Medical Center, Torrance,
California 90502 and ¶ UCLA School of Medicine,
Los Angeles, California 90024
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ABSTRACT |
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Fatty acid cycling by chain shortening/elongation
in the peroxisomes is an important source of fatty acids for membrane
lipid synthesis. Its role in the homeostasis of nonessential fatty
acids is poorly understood. We report here a study on the cycling of saturated fatty acids and the effects of troglitazone in HepG2 cells in
culture using [U-13C]stearate or
[U-13C]oleate and mass isotopomer analysis. HepG2 cells
were grown in the presence of 0.7 mmol/liter
[U-13C]stearate or [U-13C]oleate, and in
the presence and absence of 50 µM troglitazone for
72 h. Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal -oxidation of uniformly 13C-labeled stearate (C18:0) and
oleate (C18:1) resulted in chain shortening and produced uniformly
labeled palmitate (C16:0) and palmitoleate (C16:1). In untreated cells,
16% of C16:0 was derived from C18:0 and 26% of C16:1 from C18:1 by
chain shortening. Such contributions were significantly increased by
troglitazone to 23.6 and 36.6%, respectively (p < 0.001). Desaturation of stearate contributed 67% of the oleate, while
reduction of oleate contributed little to stearate (2%). The
desaturation of C18:0 to C18:1 was not affected by troglitazone. Our
results demonstrated a high degree of recycling of C18:0 and C18:1 to
C16:0 and C16:1 through chain shortening and desaturation. Chain
shortening was accompanied by chain elongation in the synthesis of
other long chain fatty acids. Troglitazone specifically increased
recycling by peroxisomal
-oxidation of C18 to C16 fatty acids, and
the interconversion of long chain fatty acids was associated with
reduced de novo lipogenesis.
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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.
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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
Spectral data can further be processed to provide information on the distribution of labeled mass isotopomer (mi) and molar enrichment (ME) using the method of Lee et al. (9) that corrects for the contribution of derivatizing agent and 13C natural abundance to the mass isotopomer distribution of the compound of interest. The resultant mass isotopomer distribution represents the fraction of molecules containing 0, 1, 2, 3, ... 13C substitutions and is expressed as a fraction of the total number of molecules. The observed number of 13C atoms incorporated per molecule is the ME. ME is the stable isotope counterpart of specific activity of radioisotopes and is expressed in units of atoms of isotope per molecule. It is calculated from the mass isotopomer distribution (mi) by the formulaDetermination 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).
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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.
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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.
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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).
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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.
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FOOTNOTES |
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* This work was supported by National Institutes of Health United States Public Health Service Grants PO1-CA 42710, MO1-RR 00425, and RO1-DK46353.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Harbor UCLA Research and Education Institute, 1124 W. Carson St., Torrance, CA 90502. Tel.: 310-222-6729; Fax: 310-533-0627; E-mail: lee{at}gcrc.humc.edu.
The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor-
; GC, gas chromatography; MS, mass
spectrometry; ME, molar enrichment.
2 A molecule is "unlabeled" when it is made up of atoms of natural isotopes, i.e. with 13C, 2H, and 18O in their natural abundances. "Unlabeled" and "unenriched" are used interchangeably.
3 The common practice is to normalize the intensities of all peaks to the base peak or the peak with the most intensity, which is set to 100%.
4
For example, the fraction of molecules of
natural stearate with mass of 298 and 299 are 0.824 and 0.176. The
average mass is given by the sum of the products 298 × 0.824 and
299 × 0.176, or 245.47 + 52.71 = 298.18. Since there are 18 carbons in stearate, the enrichment in 13C is (298.18 298)/18 or 1%, which is the natural abundance observed.
5 For example, the fractions of palmitate from HepG2 cells incubated with [U-13C]stearate having mass of 285, 286, and 287 (corresponding to m + 15, m + 16, and m + 17) are as follows: 0.094, 0.141, and 0.003. Thus the fraction of palmitate from chain shortening is 0.094 + 0.141 + 0.003 = 0.238 or 23.8%.
6
m/z 265 is the result of loss of
-OCH3 (296 31 daltons) and m/z 264 from the
loss of -HOCH3 (296
32 daltons). The corresponding M
31 and M
32 ions are also observed for palmitoleate.
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REFERENCES |
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