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J. Biol. Chem., Vol. 279, Issue 40, 41302-41309, October 1, 2004

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Coordination of Peroxisomal -Oxidation and Fatty Acid Elongation in HepG2 Cells^*

Derek A. Wong, Sara Bassilian, Shu Lim, and Wai-Nang Paul Lee

From the Department of Pediatrics, Harbor-UCLA Research and Education Institute, UCLA School of Medicine, Torrance, California 90502

Received for publication, June 17, 2004 , and in revised form, July 22, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 
A major product of mitochondrial and peroxisomal -oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal -oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of -oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-¹³C₁₈]stearate or [U-¹³C₁₆] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mM [U-¹³C₁₈]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mM [U-¹³C₁₆]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal -oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 
-Oxidation of long chain fatty acids in various tissues such as liver, heart, and muscle is known to occur in both mitochondria and peroxisomes. The integration of mitochondrial and peroxisomal -oxidation leads to optimal generation of high energy phosphate bonds and the basic two-carbon units (acetyl-CoA) for biosynthesis. These -oxidation-dependent processes are affected by internal factors such as the expression of specific ligand-activated transcription factors, peroxisome proliferators (1), and external factors such as nuclear receptor ligands and specific substrate concentrations (2). The relative contribution of mitochondrial and peroxisomal -oxidation of long chain fatty acids in isolated tissues is largely unknown. Previous methods of estimating mitochondrial and peroxisomal -oxidation of palmitate and stearate relied on the use of specific 1-¹⁴C- or 1-¹³C-labeled long chain fatty acids as precursors. Because labeled CO₂ and acid-soluble organic acids are products common to all forms of -oxidation, such methods cannot quantitatively distinguish between mitochondrial and peroxisomal -oxidation. Moreover, studies based on these methods cannot differentiate chain shortening from complete oxidation (3).

Recently, we have developed several mass isotopomer methods to estimate acetyl-CoA enrichment in the mitochondria and cytosol using uniformly labeled stearate (4, 5). This study was designed to investigate the role of peroxisomal -oxidation in very long chain fatty acid synthesis. To accomplish this, we extended our previous analysis to determine the contribution of uniformly labeled [U-¹³C₁₆]palmitate (u-palmitate)¹ and [U-¹³C₁₈]stearate (u-stearate) to the acetyl-CoA pool used in very long chain fatty acid synthesis in HepG2 cells. We estimated the acetyl-CoA enrichments used in de novo lipogenesis (cytosolic pool) in the tricarboxylic acid cycle (as reflected by the glutamate pool) and in chain elongation of stearate (peroxisomal pool). Combining these results with analysis of chain shortening and chain elongation, we demonstrated that -oxidation of palmitate and stearate is compartmentalized and differentially regulated, resulting in disparate acetyl-CoA enrichment in these compartments. Acetyl-CoA derived from peroxisomal chain shortening of stearate is a significant source of carbon units for very long chain fatty acid synthesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 
Cell Culture Experiments—Human hepatoma cell line HepG2 (American Type Culture Collection, HB-8065) was maintained in 75-cm² tissue culture flasks in Dulbecco's modified Eagle's medium augmented with 20% (v/v) fetal bovine serum, 1000 mg/liter glucose, and 1% penicillin (100 units/ml)/streptomycin (100 µg/ml) plus amphotericin B (0.25 µg/ml) at 37 °C in a humidified atmosphere containing 5% CO₂. When the cultures were 90% confluent (2.5 x 10⁶ cells/plate), the medium was removed, and monolayers were washed 1 time with phosphate-buffered saline. The cells were then incubated in media enriched with uniformly labeled [¹³C₁₆]palmitic acid or [¹³C₁₈]stearic acid (Spectra Stable Isotopes, catalog numbers 52339 and 52349) at concentrations of 0, 0.1, 0.2, and 0.5 mmol/liter. These compounds were tested for purity by GC/MS and found to have no traces of longer or shorter chain fatty acids. Fatty acids were dissolved in alcohol and added to media (total alcohol equalized in all flasks to 0.1% by volume). Four flasks were prepared for each fatty acid at each enrichment and two for control conditions. Media was changed at 0, 24, and 48 h, and cells were harvested from the plates at 72 h.

Lipid Extractions—Lipid extractions were performed using methods described by Lowenstein et al. (6). Briefly, cells from each plate were harvested, and heptadecanoic acid (C17:0) and tricosanoic acid (C23:0) were added as internal standards for fatty acids. The mixture was saponified with 200 µl of 30% KOH-ethanol (1:1 v/v) at 70 °C overnight. The aqueous phase was acidified, and fatty acids were extracted in petroleum ether and dried under a stream of nitrogen. Fatty acids were methylated with 0.5 N HCl in methanol (Supelco) for GC/MS analysis.

GC/MS Analysis; Lipids—GC/MS analysis was performed on a Hewlett Packard model 5973 Selective Mass Detector connected to a Model 6890 gas chromatograph using electron impact ionization and selected ion monitoring to follow specific ions. Fatty acid esters were separated using glass capillary column bpx 70 (SGE) measuring 30 m x 250 µm inner diameter. The GC conditions for fatty acids were: carrier gas (helium) flow rate, 1 ml/min; injector temperature, 250 °C; temperature programming, 140 to 230 °C at 5 °C/min. In the following sections, we use m0 or m+0 interchangeably to indicate the base peak and m14 or m+14 to indicate the isotopomer with a mass shift of 14 daltons of myristate. This nomenclature is applied to all fatty acids and metabolites studied. The ions monitored were: myristate (C14:0) m0 cluster, m/z 241–248, and m14 cluster, m/z 253–260; palmitate (C16:0) m0 cluster, m/z 269–76, and m16 cluster, m/z 284–303; heptadecanoate (C17:0), m/z 284; stearate (C18:0), m0 cluster, m/z 297–304, and m18 cluster, m/z 312–318; oleate (C18:1) m0 cluster, m/z 264–269, and m18 cluster, m/z 277–284; arachidate (C20:0) m0 cluster, m/z 325–331, and m18 cluster, m/z 340–348; behenate (C22:0) m0 cluster, m/z 353–360, and m18 cluster, m/z 368–376; tricosanoate (C23:0), m/z 368, lignocerate (C24:0), m0 cluster, m/z 381–388, and m18 cluster, m/z 396–404.

Glutamate Extractions—Tissue culture medium was first treated with 6% perchloric acid and centrifuged to remove protein. The supernatant was neutralized with KOH and then passed through 5-cm³ Dowex-50 columns. Amino acids were eluted from the column with 10 ml of 2 N ammonium hydroxide followed by air-drying. To separate glutamine from glutamate, amino acids were dissolved in water and passed on 5-cm³ Dowex-1 columns. Glutamine was washed off with 15 ml of water. Glutamate was eluted with 15 ml of 0.5 N acetic acid and air-dried. Glutamate was derivatized using the method of Leimer et al. (7). Glutamate was heated to 100 °C for 2 h in 100 µl of HCl in butanol (Regis). Butanol and HCl were removed under a stream of nitrogen, the residue was dissolved in methylene chloride, and 100 µl of trifluoroacetic anhydride was added to complete the reaction. The trifluoroacetamide butyl ester of glutamate was dissolved in methylene chloride for GC/MS analysis.

GC/MS Analysis; Glutamate—A Hewlett-Packard model 6890 gas chromatograph connected to a model 5973 mass spectrometer was used. The capillary column was HP5 measuring 30 m x 250 µm. The GC conditions were as follows: injector temperature, 250 °C; temperature programming was 205 to 215 °C at 3 °C/min. Selected ion monitoring was used to follow specific ions. Under EI conditions, ionization of the trifluoroacetamide butyl ester of glutamate gives rise to two fragments, m/z 198 and m/z 152, corresponding to C2-C5 and C2-C4 of glutamate, respectively (8).

Data Analysis; Fatty Acids—Mass isotopomer distribution was determined using the method of Lee et al., which corrects for the contribution of derivatizing agent and ¹³C natural abundance to the mass isotopomer distribution of the compound of interest (9). The calculated mass isotopomer distribution is expressed as molar fractions (m0, m1, m2, m3, etc.), which are the fractions of molecules containing 0, 1, 2, 3,... ¹³C substitutions, respectively. Data reduction and regression analyses were performed using the computer software Microsoft Excel® version 5.0 and Wolfram Research Mathematica® version 5.0.1.

Determination of Precursor Enrichment—-Oxidation of palmitate and stearate generates acetyl-CoA, which is subsequently used as a precursor for de novo lipogenesis, chain elongation, and energy production via the tricarboxylic acid cycle. Thus, -oxidation can be assessed through the determination of acetyl-CoA enrichment in the different synthetic products.

Acetyl-CoA Enrichment for de Novo Synthesis—The enrichment of the cytosolic acetyl-CoA pool used in de novo fatty acid synthesis was determined from the mass isotopomer distribution in 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 been previously shown to be a binomial distribution (10, 11). Thus, the acetyl-CoA enrichment may be obtained from the consecutive mass isotopomer ratio m4/m2 = (n – 1)p/q = 3.5p/q, where n is the number of acetyl units in palmitate = 8, p is the enrichment of [1,2-¹³C₂]acetyl-CoA, q is the unenriched acetyl-CoA, and p + q = 1.

Acetyl-CoA Enrichment from Mitochondrial -Oxidation—[1,2-¹³C₂]acetyl-CoA produced from -oxidation combines with oxaloacetate to form citrate in the mitochondria. The citrate participates in the tricarboxylic acid cycle, eventually forming [-4,5-¹³C₂]ketoglutarate and glutamate (12). The enrichment of the m2 component of the C2-C5 fragment generally reflects tricarboxylic acid cycle activity (13, 14). However, it is well known that contribution from unlabeled glutamate from the cells in culture with glutamine-containing medium can substantially dilute the glutamate enrichment (15). Thus, the enrichment of the [4,5-¹³C₂]glutamate is the lower limit of mitochondrial acetyl-CoA pool.

Acetyl-CoA Enrichment for Chain Elongation—Enzymes involved in chain elongation from palmitate and stearate to longer chain fatty acids are located in the endoplasmic reticulum, mitochondria, and peroxisomes (16). Chain elongation of uniformly labeled palmitate and stearate with [1,2-¹³C₂]acetyl-CoA produces characteristic m+16 and m+18 clusters that can be used to determine the acetyl-CoA enrichment according to the rules of combination of two labeled precursors. The probability of forming m+16 stearate from u-palmitate is the joint probability of combining m+0 acetyl-CoA and m+16 palmitate. Similarly the probability of finding m+18 stearate is the joint probability of combining m+2 acetyl-CoA and m+16 palmitate. Thus, the ratio of m+2 to m+0 acetyl-CoA for chain elongation is given by the ratio of m+18 to m+16 in stearate in u-palmitate experiments and m+20 to m+18 in arachidate in u-stearate experiments. The enrichment of [1,2-¹³C₂]acetyl-CoA for each step of chain elongation (C16 to C18 in u-palmitate experiments and C18 to C20 in u-palmitate and u-stearate experiments) was determined using the method described in the appendix, which corrects for the fact that ¹³C enrichment in u-fatty acids is less than 100%.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 
Fatty acid analysis of cell pellets shows two clusters in each spectrum, one "light" cluster representing newly synthesized and preexisting fatty acids and one "heavy" cluster representing fatty acids derived from uniformly labeled precursors (Fig. 1). u-palmitate-enriched cells have a heavy cluster consisting of a very high m+16 peak and a smaller m+15 peak arising from small amounts of ¹²C (<2%) in u-palmitate (m/z 285–286, Fig. 1b). Similarly, u-stearate-enriched cells have a heavy cluster with a large m+18 peak and smaller m+17 peak (m/z 315–316, Fig. 1e). Chain shortening of added u-stearate produced a small percentage of m+16 palmitate (m/z 286, Fig. 1e), whereas chain elongation of added u-palmitate produced a much larger percentage of m+18 stearate (m/z 314, Fig. 1c). A large fraction of C20:0 is derived from elongation of C16:0 (m/z 342 in u-palmitate cell cultures, Fig. 1d) and C18:0 (m/z 344 in u-stearate cell cultures, Fig. 1g).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Normalized spectra of saturated fatty acids from cells incubated with u-palmitate (a–d) or u-stearate (e–h). These spectra show the presence of the heavy clusters from chain elongation or chain shortening of these perlabeled fatty acids. Charts are labeled by fatty acids; for example, uc16c14 represents the spectrum of C14:0 in u-palmitate-enriched cells. Long chain fatty acid spectra from chain elongation of u-palmitate and u-stearate showed different isotopomer distribution in the heavy cluster because of differences in the acetyl-CoA used in the chain elongation process.

View this table: [in this window] [in a new window]   TABLE II Acetyl-CoA enrichment contributed by -oxidation in different cell compartments Enrichment is expressed as mole percent. Average ± S.D. of results (n = 4) are presented.

Analysis of the palmitate and stearate spectra in these experiments allows differentiation between preexisting, newly synthesized, added, and chain-shortened or elongated products (Figs. 2 and 3). Table III shows direct measurements taken from these spectra, which are used as the basis for subsequent calculations. u-palmitate enrichments of 0.1–0.5 mM resulted in a heavy ion percentages of 43.45–82.24%, whereas u-stearate enrichment at the same concentrations resulted in a C18 distribution with 67.67–93.32% heavy ions. De novo lipogenesis introduces m+2 and m+4 ions within the light clusters, which can be used to determine the fraction of newly synthesized molecules (FNS). The FNS for each experimental condition is minimally affected by the added fatty acids.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Analysis of palmitate spectra. The mass spectrum of palmitate is the composite of spectra of palmitate molecules from several sources. The final spectrum of palmitate is the weighted average of spectra of molecules from these sources according to their relative abundances (concentrations).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Analysis of stearate spectra. The mass spectrum of stearate is the composite of spectra of stearate molecules from several sources. The final spectrum of stearate is the weighted average of spectra of molecules from these sources according to their relative abundances (concentrations).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Contribution of u-palmitate (uC16 C18:1) and u-stearate (uC18 C18:1) to isotopomer enrichment in oleate. The contribution is expressed as the fraction of ions in the heavy clusters as percent of total ions of the oleate spectrum.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

Based on the disparate acetyl-CoA enrichments in different pools summarized in Table II and the differences between enrichments of acetyl-CoA from u-stearate and u-palmitate treatments, we propose a model with three specific functional compartments: peroxisomal, mitochondrial, and lipogenic/cytosolic (Fig. 5). Added palmitate or stearate enters the cell and undergoes either mitochondrial or peroxisomal -oxidation. Mitochondrial -oxidation of these fatty acids produces acetyl-CoA, which rapidly diffuses to the lipogenic compartment and is incorporated into newly synthesized fatty acids. It is in the peroxisomal compartment that the metabolic fates of stearate and palmitate differ. The acetyl-CoA generated from chain shortening of stearate enriches the peroxisomal compartment. Chain shortening of stearate and longer chain fatty acids is coupled to very long chain fatty acid synthesis, whereas the peroxisomal chain shortening of palmitate has no effect on the same processes. The net effect of this reaction is that the total number of acyl-CoA molecules is conserved, and the fatty acid composition is changed. For every two molecules of stearate entering the peroxisome, one is chain-shortened to palmitate, and the acetyl-CoA generated condenses with the second stearate molecule to form behenate. In theory, the peroxisomal compartment controls the rates of very long chain fatty acid degradation, stearate chain shortening, and very long chain fatty acid synthesis.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Compartmentalization of fatty acid metabolism. The results of mass isotopomer distribution analysis support a functional model consisting of the mitochondrial, lipogenic, and peroxisomal compartments. These compartments are accessible to free fatty acids as indicated by arrows going to various fatty acid pools, and their metabolism is regulated as indicated by unidirectional pathways. Because synthesis and degradation may not occur in the same physical space, these pathways are indicated by different arrows as shown in the peroxisomal compartment for chain shortening and chain elongation

Although most early studies of peroxisomal metabolism focused on very long chain and branched chain fatty acid oxidation, there is increasing evidence that chain shortening of long chain fatty acids plays an important role in a variety of physiologic processes. In this study, peroxisomal chain shortening of stearate was coupled to a 4–10-fold increase in C20:0 concentration. In fetal rabbit lung type II cells, palmitate derived from stearate chain shortening is preferentially incorporated into phosphatidylcholine (19). Recent studies using uniformly labeled oleate and palmitate showed a substantial contribution of chain shortening to the malonyl-CoA pool of the heart (17). Human acyl-CoA oxidase 1 has highest activity on medium to long chain substrates, with relative activity of only 6% toward C24:0 compared with 90–100% activity toward C8:0, C12:0, and C16:0 (20). Although this difference may be related to the more general role of the peroxisome in fatty acid oxidation in pre-mitochondrial ancestral organisms, the subsequent lack of evolution toward a more specific enzyme argues for a strong pressure to preserve chain-shortening capabilities. The results of our present study are entirely consistent with this view.

The use of C18-C20 chain elongation and other mass isotopomer analysis is the first quantitative method to determine peroxisomal acetyl-CoA enrichment (and therefore peroxisomal -oxidation). The method should prove useful in the study of both peroxisomal oxidation and very long chain fatty acid synthesis in both cell culture and animal experiments. Our present study did not reveal the fate of acetyl-CoA in other situations, such as peroxisomal palmitate chain shortening. Further studies are necessary to increase our understanding of acetyl-CoA compartmentalization and regulation of chain elongation, particularly in the diagnosis and treatment of disease due to inborn errors of fatty acid metabolism.

	APPENDIX

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 
Uniformly labeled palmitate and stearate have spectra which are shifted by 16 and 18 daltons from their unlabeled ions. [1,2-¹³C]Acetyl-CoA is produced both by oxidation of u-palmitate and by chain shortening of u-stearate to u-palmitate. u-palmitate and u-stearate are elongated with this labeled acetyl-CoA as well as unlabeled acetyl-CoA. Because the ¹³C enrichment in u-palmitate and u-stearate is not 100%, the impurity from ¹²C resulted in the presence of m+14 and m+15 in u-palmitate and m+16 and m+17 in u-stearate. The presence of these isotopomers contributed to the complexity of data reduction.

The effective acetyl-CoA enrichment in the reaction C(X:0) + acetyl-CoA C(X + 2:0) is determined from the shifted distributions of C(X:0) and C(X + 2:0). The equation is: F·E = G, where F is the shifted distribution matrix of C(X:0), representing a fatty acid of length X carbons before elongation, E is the acetyl-CoA enrichment vector, and G is the shifted distribution vector of the elongation product fatty acid with length X + 2 carbons. The E vector is determined by linear regression. Because both the fatty acid and its elongation product are derivatized shifted spectra, there is no need to correct for the derivative or natural abundance before regression. An example calculation from the elongation of C18 to C20 is given in Table VI.

Chain elongation of u-palmitate or u-stearate to longer chain fatty acids such as behenate (C22:0) and lignocerate (C24:0) also resulted in a shifted distribution, with isotopomers at m + 16, m + 18, m + 20, m + 22, and m + 24. Because C18, C20, and C22 can all participate in chain elongation, the elongation of palmitate and stearate to long chain fatty acids by the addition of more than one acetyl unit cannot be explained by a simple binomial chain elongation model (4).

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK56090–04 (to W.-N. P. L.). The GC/MS facility is supported by Public Health Service Grants P01-CA42710 (to the UCLA Clinical Nutrition Research Unit, Stable Isotope Core) and M01-RR00425 (to the General Clinical Research Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Derek Wong is supported by Public Health Service Training Grant GM-08243.

To whom correspondence should be addressed: Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502. Tel.: 310-222-6729; Fax: 310-222-3887; E-mail: lee{at}gcrc.rei.edu.

¹ The abbreviations used are: u-palmitate, [U-¹³C₁₆]palmitate; u-stearate, [U-¹³C₁₈]stearate; GC/MS, gas chromatography/mass spectroscopy; FNS, fraction of newly synthesized molecules.

² Another potential source of dilution of the mitochondrial pool, the conversion of ethanol to acetate via the microsomal ethanol oxidizing system pathway, was shown to have minimal impact (unpublished results).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

 

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