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Vol. 41, No. 3, March 1997 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages597-607 METABOLIC FATE AND EFFECTS OF SATURATED AND UNSATURATED FATTY ACIDS IN HEP2 HUMAN LARYNX TUMOR CELLS Alison Colquhoun ~ and Rui Curl, Department of Physiology and Biophysics, Institute of Biomedical Sciences l, University of S~o Paulo, Brazil Received December 18, 1996 Previous studies have reported the presence of carnitine palmitoyltransferase 1 and II in tumor cells and the inhibitory effects of fatty acids on cell proliferation. The present work considered the metabolic fate of [14C] or [3HI-labeled fatty acids and their effects on cellular metabolism in Hep2 human larynx tumor cells.The rate of uptake of acetate was 45% of that of myristate, paimitate, oleate, iinoleate and arachidonate. However, acetate was rapidly metabolized within the cell as seen by its low rate of accumulation as non-esterified fatty acid, <5% of that of the other fatty acids. The incorporation of fatty acids into neutral lipid fractions showed palmitate and oleate primarily entered the phospholipid fraction, while linoleate and arachidonate entered equally the phospholipid and triacylglycerol fractions. Palmitate and oleate were oxidized to ~4CO2 at higher rates than linoleate and arachidonate, with arachidonate being the least oxidized of the unsaturated fatty acids. Acetate was oxidized at 10-30 fold higher rates than the other fatty acids. Palmitate, oleate, linoleate and arachidonate all had significant inhibitory effects on the rate of glucose utilization by Hep2 cells, ranging from 25-38% inhibition and were found to inhibit cell proliferation by 17-73%. These findings suggest that certain fatty acids not only play a structural role in cellular metabolism, but may also have a potential regulatory role in the glycolytic pathway of Hep2 cells. Key words:- Fatty acids; glycerolipids; phospholipids; carnitine palmitoyltrausferase; ~-oxidation; glucose metabolism; tumor cell proliferation INTRODUCTION Cultured rat hepatocytes (1) and cardiomyocytes (2) have been reported to oxidize fatty acids of varying chain length at differing rates. Isolated rat liver mitochondria have also been found to oxidize 18-carbon fatty acids at differing rates dependent upon the degree of unsaturation, with higher oxidative capacity for a-linolenate than for oleate (3). The ratelimiting regulatory enzyme of I~-oxidation in liver, carnitine palmitoyltransferase I (CPT 1), Corresponding author: Alison Colquhoun, Departamento de Fisiologia e Biofisica, Instituto de Ci~ncias Biom6dicas I, Universidade de $5,o Paulo, Silo Paulo, CEP 05508-900, SP, Brasil. Tel. 011 818 7225/Fax 0l I 818 7402; E-mail : [email protected] Abbreviations: BSA, bovine serum albumin; CPT I / II, carnitine palmitoyltransferase I / II; CoA, coenzyme A; M/DAG, mono/diacylglycero[; HCIO4, perchloric acid; PL, phospholipid; TAG, triacylglycerol. 1039-9712/97/030597-1 I $05.00/0 597 C'r 9 1997 by Academic Press Auslrulm All rights oJ reproduction in any form reserved Vol. 41, No, 3, 1997 BIOCHEMISTRYGnd MOLECULAR BIOLOGYINTERNATIONAL has substrate specificity for fatty acids, with higher activity for ot-linolenate than for oleate or palmitate (4,5). Hence, in rat liver the substrate specificity of CPT I regulates the rate of oxidation of individual fatty acids within the mitochondrion. Fao rat hepatoma cells have been reported to oxidize oleate at significantly lower rates than hepatocytes and to possess a form ofCPT I which is highly sensitive to inhibition by malonyl CoA (6). Fatty acids are also used as structural components within the cell and are incorporated into phospholipids and neutral lipids at rates dependent upon the individual fatty acid. Hepatocytes have been shown to incorporate fatty acids preferentially into triacylglycerols, with considerably less being incorporated into phospholipids, TAG/PL ratios ranging from 2.7-3.6 in rat and hamster hepatocytes, respectively (6,7). Incorporation of fatty acids into tumor cells has been reported to have preference for phospholipids in human breast tumor cells (8) and Fao rat hepatoma, TAG/PL ratio of 0.71, (6), reflecting the high requirement for phospholipids for membrane biosynthesis in these rapidly dividing cells. Finally, fatty acids are also known to influence tumor cell proliferation, with eicosapentaenoate and docosahexaenoate being the most commonly cited inhibitors (8,9,10). Less is known about the fatty acid oxidative capacity of human tumor cells than those from rodents, human tumor cells having recently been shown to possess active mitochondrial CPT I and II (11) and, hence, the capacity to oxidize long-chain fatty acids within the mitochondrion. However, the specificity of CPT I in human tumor cells for fatty acids has not been determined nor have the effects of fatty acids upon cellular energy metabolism. The aim of the present work was, therefore, to investigate fatty acid metabolism in a human tumor cell line taking into consideration four main areas :- (1) the specificity of fatty acid uptake and intracellular accumulation in its non-esterified form ; (2) the relative rates of incorporation of individual fatty acids into intracellular glycerolipids ; (3) the specificity of CPT I for fatty acids, determined indirectly by oxidation of [14C]-labeled fatty acids to 14CO2 ; (4) the effects of fatty acids upon cellular glucose metabolism and proliferation. MATERIALS AND M E T H O D S Cell Culture - Hep2 human larynx tumor cells were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK, and grown in Minimum Essential medium supplemented with 10% fetal calf serum and antibiotics (penicillin 50U/ml, streptomycin 50gg/ml). Cells in the exponential phase of growth were used throughout the study, growing in 25cm2 tissue culture flasks in a humidified atmosphere of 5% CO2:95% air at 37~ Measurement of fatty acid oxidation - Hep2 cells were incubated with [1-14C]-labeled fatty acids for a period of 6 hours, gassed (5%CO2:95% air) and sealed in 25cm2 flasks adapted to contain a central well plus filter paper for subsequent collection of ~4CO2. At the 598 Vol. 41, No, 3, 1997 BIOCHEMISTRYond MOLECULAR BIOLOGYINTERNATIONAL end of the incubation period 0.2mI of phenylethylamine/methanot (1:1, by vol) was injected into the central well, followed by injection of 0.2ml HCIO4 (30%, by vol) into the culture medium to release the 14CO2. The sealed flasks were shaken for 1 hour at. room temperature to trap the 14CO2 on the filter. Radioactivity was determined by scintillation counting. Five or six separate experiments were performed for each fatty acid studied. Determination of fatty acid incorporation into cellular lipids - Four separate experiments were performed for each fatty acid studied, yielding similiar results, data from which have been pooled. All the fatty acids used were [1-14C]-labeled with the exception of [U-14C]-palmitate and [9,10-3H]-myristate. Before the experiment each cell monolayer was washed three times with serum-free medium. The experimental medium consisted of serumfree medium, 0.5gCi of radiolabeled fatty acid, fatty acid-defatted BSA complex (molar ratio 7.2:1) to give 150gM total fatty acid concentration and defatted BSA to give 0.1% total BSA concentration. Hep2 cells were incubated for 6 hours then cellular lipids were extracted by the method of Folch (t2). Solvents were evaporated to dryness under a stream of nitrogen and the dried lipid extracts dissolved in 501A chloroform/methanol (2:1, by vol). Lipid samples were loaded onto silica gel 60 glass plates and neutral lipids were separated with the solvent system hexane/diethylether/glacial acetic acid (70:30:1, by vol), with nonradioactive standards for band identification. The lipid fractions were identified by exposure to iodine vapor. Bands were cut from the plate and the radioactivity in each fraction determined by scintillation counting. Determination of glucose utilization - Hep2 cells were incubated for 6 hours in 25 cm2 flasks, at the beginning and end of which culture medium was collected for subsequent analysis of glucose content by the method of(13). Other methods - Cellular protein was determined by the method of (14). Experimental analysis - Experiments were carried out a minimum of four times, if more this is stated in the relevant text/table. Statistical significance was determined by Student's ttest, with significant difference being set at p<0.05. Materials - Culture medium and related materials were from Gibco, USA, bovine serum albumin and general chemicals were from Sigma Chemical Co., USA. [1-14C]-actetate, [9,10_3H]_myristate, [U_14C]_palmitate, [l_I4C]-oleate, [1-14C]-linoleate and [1-~4C]arachidonate were from Amersham International Ltd., UK RESULTS Fatty acid uptake - When analyzed after a 6 hour culture period, extracellular fatty acids were found to have been rapidly removed from the culture medium by Hep2 cells. The data in Table 1 represents the percentage of labeled fatty acid removed from the medium for six differing fatty acids. All fatty acids were removed at similar rates with the exception of the short-chain fatty acid, acetate. Acetate appears to be taken up by the tumor cells at approximately 45% of the rate of uptake of the other fatty acids studied. The rates of uptake were similar to those reported for hamster hepatocytes, 70-80% at 8 hours for oleate and palmitate, (7) and for human breast tumor cells MDA-MB-435, 37-72% at 6 hours for linoleate and eicosapentaenoate, (8). 599 Vol. 41, No, 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL TABLE ! Amount of fatty acid removed from the culture medium by Hep2 cells Fatty acid Fatty acid removed after 6 hours (%) Acetate 31.1 + 1.86 Myristate 70.9 + 1.3 Palmitate 69.0 + 2.7 Oleate 76.8 + 4.0 Linoleate 75.8 +4.0 Arachidonate 75.7 + 6.3 Values are the mean +_S.E.M. for six separate observations. Statistical significance : ~' - v s all other fatty acids. Of the fatty acids taken up by the cell there was a significant difference in the subsequent accumulation of non-esterified fatty acid depending on the individual fatty acid (Table 2). Acetate accumulation was lowest followed by myristate, linoleate and oleate, with the highest accumulation being that of palmitate and arachidonate. While the uptake of acetate was -45% of the rate of the other fatty acids, its accumulation within the cell was <5% of the others, thereby indicating its rapid metabolism within the cell. Rates of accumulation were similar to those seen in rat hepatocytes, expressed as nmoles/hr/mg protein: myristate (0.66); oleate (1.69) and linoleate (0.36). However, palmitate accumulated at far higher rates in hepatocytes than in Hep2 cells, with a rate of 11.85 nmoles/hr/mg protein (1). Cholesterol synthesis from fatty acids, expressed as a percentage of total fatty acid incorporation, was highest from acetate (8.0%), followed by myristate (5.3%), palmitate (4.0%), arachidonate (2.3%), linoleate (2.1%) and oleate (1.3%). Variations in cholesterol synthesis from individual fatty acids may be due to substrate specificity of this process in Hep2 cells or possibly to unsaturated fatty acid effects on ATP citrate lyase activity, linoleate, arachidonate and eicosapentaenoate having been shown to inhibit mRNA expression &this gene in hepatocytes (15). Fatty acid incorporation into intraceilular glyeerolipids - The rate of incorporation of fatty acids into the glycerolipids; phospholipids, mono- and diacylglycerols and triacylglycerols are presented in Table 3, The rate of acetate incorporation into all three classes of glycerolipid was the lowest of the six fatty acids studied. In the case of the 600 Vol. 41, No. 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL TABLE 2 Accumulation of non-esterified |1-14C] or [9,10-3H] labeled fatty acids-in Hep2 cells Non-esterified fatty acid accumulation Fatty acid nmoles/hr/mg protein Acetate 0.046 +0.02" Myristate 0.91 + 0.18 b'c Palmitate 2.93 + 0.58 a Oleate 1.46 + 0.36 Linoleate 1.33 + 0.12 ~ Arachidonate 2.39 + 0.18 Values are the mean + S.E.M. for six separate observations. Statistical significance: all other fatty acids; b _ v s palmitate; ~' - v s arachidonate; a _ vs linoleate " - vs TABLE 3 Rate of fatty acid incorporation into intracellular glycerolipids Fatty acid incorporation Fatty acid (nmoles/hr/mgprotein) M/DAG TAG PL Z Acetate 0.019 + 0 . 0 0 l 0.037 + 0.01" 0.11 + 0 . 0 4 " 0.166+0.056" Myristate 0.66 + 0.37 2.71 + 0.62 d' ~ 2.93 + 0 . 2 6 b'~'a'~ 6.30+_ 1.26 b'~'a'~ Palmitate 1.26 + 0.30 c' a, e 3.60 +_ 0.49 a' ~ 11.20 +_ 1.66 e 16.06 + 2.78 Oleate 0.36 +0.15 5.05 + 2.19 10.91 + 2 . 3 6 16.31 + 2 . 3 2 Linoleate 0.31 + 0.11 6.16 + 0.88 ' 6.11 _+0.27 ~ 1 2 . 5 7 + 1.10 e Arachidonate 0.40 + 0 . 1 0 9.89 + 0.59 9.23 _+ 0.69 19.51 + 1.14 Values are the mean _+ S E . M . for four separate observations. Statistical significance: - v s all other fatty acids; b _ v s palmitate; ~ - vs oleate; d _ v s linoleate; e _ v s arachidonate M / D A G - mono/diacylglycerols; T A G - triacytglycerols; PL - phospholipids; E - sum o f M / D A G + T A G + PL mono/diacylglycerol fraction all the fatty acids were incorporated at similar rates with the exception o f palmitate. However, in both the triacylglycerol and phospholipid fractions the fatty acids fell into three groups. Acetate was incorporated at low rates into both fractions, but predominantly into the phospholipid fraction, with a T A G / P L ratio o f 0.34. Patmitate and oleate were incorporated predominantly into the phospholipid fraction, with T A G / P L ratios o f 0.32 and 0.46, respectively. Myristate, tinoleate and arachidonate were each 601 Vol. 41, No, 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL incorporated equally into the phospholipid and triacylglycerol fractions, with TAG/PL ratios of 0.92, 1.01and 1.07, respectively. By calculating the sum of the three fractions, the rate of incorporation into glycerolipids was determined. Acetate was incorporated into glycerolipids at <3% of the rate of any other fatty acid. This was followed by myristate, which was incorporated at 40%, 39%, 50% and 32% of that of palmitate, oleate, linoleate and arachidonate, respectively. Of the unsaturated fatty acids, arachidonate and oleate were incorporated at higher rates than linoleate and at rates equal to that of palmitate. Unlike Hep2 cells, hepatocytes incorporate far more fatty acid into the TAG fraction than the phospholipid fraction, TAG/PL ratio of between 2.7-3.6 for palmitate and oleate in rat and hamster hepatocytes (6,7). 13-Oxidation of fatty acids - The rates of oxidation of fatty acids by Hep2 were clearly seen to fall into three categories (Table 4).The least oxidized were polyunsaturated arachidonate and linoleate, then monounsaturated oleate, then saturated palmitate. However, the fatty acid most readily oxidized was acetate, this being oxidized at rates almost 10-fold, 20-fold and 30-fold greater than palmitate/oleate, linoleate and arachidonate, respectively. Rat hepatocytes oxidized palmitate, oleate and linoleate at similar rates, 2.40-3.30 nmoles/hr/mg protein (1) while Fao rat hepatoma cells oxidized oleate at lower rates than normal hepatocytes (6). Rat cardiomyocytes oxidized oleate at similar rates to that of hepatocytes (2.58nmoles/hr/mg protein) and docosahexaenoate at lower rates (1.46 nmoles/hr/mg protein) (2). The oxidation of the long-chain, polyunsaturated fatty acid docosahexaenoate at low rates by cardiomyocytes is consitent with the finding that the long-chain, TABLE 4 Utilization of [14C]-labeled fatty acids via mitochondrial J3-oxidation Fatty acid 14C02production nmoles/hr/mg protein Acetate 4.23 _+0.814 Palmitate 0.479 + 0.1 l Oteate 0.481 + 0.14 Linoleate 0.203 + 0.02 b Arachidonate 0.128 + 0.03 b' c Values are the mean + S.EM. for five or six separate observations. Statistical significance: a _ vs all other fatty acids; b _ vs palmitate; c _ vs oleate 602 Vol. 41, No. 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL polyunsaturated fatty acid arachidonate was oxidized at low rates by Hep2 cells. In the present study Hep2 oxidized fatty acids at significantly lower rates than that of rat hepatocytes, cardiomyocytes or hepatoma cells. Glucose utilization and fatty acids - Glucose utilization from the culture medium was determined for the six individual fatty acids (Table 5) and in the absence of added fatty acid. In the presence of both acetate and myristate glucose utilization was unaltered. On the other hand, glucose utilization was significantly reduced when cells were exposed to either palmitate, oleate, linoleate or arachidonate. DISCUSSION The fatty acids used in the present study are readily extracted from the culture medium by Hep2 human tumor cells. While acetate is taken up by the cells at only 45% of the rate of the other fatty acids it accumulates at a much lower rate, <5%, as intracellular non-esterified fatty acid. Interestingly, the degree of unsaturation of the fatty acid does not have a direct effect on its accumulation rate, since arachidonate and palmitate accumulate at higher rates than oleate and linoleate. Therefore, subsequent utilization o f the fatty acid after removal from the culture medium does not appear to be governed by either the chain length or the degree ofunsaturation of the individual fatty acid in these cells. The rate of incorporation into the glycerolipid fraction showed that all fatty acids were distributed into the mono/diacylglycerol fraction at similar rates with the exception of palmitate. This may reflect a preference for saturated fatty acids in the initial acylation of the TABLE 5 Rate of glucose utilization by Hep2 in the presence of differing fatty acids Fatty acid Glucose utilization Otmoles/hr/mgprotein) None 311.5 + 28.5 Acetate 300.0 + 16.4 Myristate 304.7 + 13.3 Palmitate 232.7 + 16.6 a Oleate 193.1 + 17.1" Linoleate 207.7 +_ 22.3" Arachidonate 217.3 + 30.2" Values are the mean + S.E.M for ten separate observations. Statistical significance : ~ - vs no added fatty acid 603 Vol. 41, No, 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 1-position of glycerol-3-phosphate, catalysed by glycerol-3-phosphate acyltransferase (5). Distinct differences were found for unsaturated and saturated fatty acid incorporation in the process of triacylglycerol synthesis, The rate of arachidonate incorporation was the highest of all the fatty acids studied, followed by linoleate and oleate. The saturated palmitate and myristate were incorporated at lower rates, with acetate incorporated at the lowest rate of all the fatty acids studied. Unsaturated fatty acids thus appear to be the preferred substrates over saturated fatty acids for triacylglycerol synthesis, suggesting diacylglycerol acyltransferase may have a higher specificity for unsaturated fatty acids in Hep2 cells. In the phospholipid fraction specificity was also seen for incorporation. The fatty acids palmitate, oleate and arachidonate were preferentially incorporated in comparison with linoleate. Myristate was incorporated at considerably lower rates than all the long-chain fatty acids suggesting it may not be a major component of the phospholipid fraction of these cells. In conclusion, the major components of the glycerolipid fraction of Hep2 cells in this study were the fatty acids arachidonate, oleate, palmitate and, to a lesser degree, linoleate. The knowledge of which fatty acids are more readily incorporated into the structural components of Hep2 begs the question of "which fatty acids are also substrates of the 13oxidation pathway?". Hep2 cells have been previously shown to possess active mitochondrial carnitine palmitoyltransferase I and II (11), indicating that they have the capacity to process acyl CoA's into the mitochondrion for subsequent oxidation. The oxidative rates of palmitate, oleate, linoleate and arachidonate, all good substrates for structural components of the cell, were found to be markedly less than that of acetate (10-30 fold), a poor substrate for structural components. It is also significant that the least oxidized unsaturated fatty acid, arachidonate, is the fatty acid incorporated into glycerolipids at the highest rate. Hence, Hep2 fatty acid oxidation, possibly via the substrate specificity of CPT I, can discriminate between both the chain length and the degree of unsaturation of fatty acids, with oxidation in the order, acetate > palmitate = oleate > linoleate > arachidonate. The variable rates of oxidation of fatty acid substrate led to the investigation of their individual effects on another energy producing pathway, that of aerobic glycolysis, which is of great importance to the tumor cell. Glucose utilization from the culture medium was used as a reflection of the glycolytic rate of the proliferating cells. Surprisingly, the most readily oxidized fatty acid, acetate, had no effect upon glucose utilization as was the case for myristate, the rate of oxidation of which was not determined in the present work. However, palmitate and oleate, which were moderately oxidized, had inhibitory effects upon glucose 604 Vol. 41, No. 3, 1997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL utilization equal to those of the less oxidized linoleate and arachidonate. Hence, CPT- I specificity for fatty acid substrate does not appear to be involved in the altered glucose utilization rates of Hep2. The nature of the inhibition of glucose utilization has not yet been identified but cannot be linked to the rate of acetyl CoA production within the cell as acetate had no inhibitory effect. Similarly, it is unlikely that simple incorporation of fatty acid into the cell can be the cause as myristate, which is readily taken up by the cell, also had no inhibitory effect. The differing TAG/PL ratios of the fatty acids studied also showed no direct link with inhibition of glucose utilization, therefore it may be that altered membrane composition is not involved, indeed over a 6 hour period this is unlikely to be the case, even though Hep2 had a doubling time of only -10 hours under these experimental conditions. One of the few putative links identified was the intracellular non-esterified fatty acid accumulation which was considerably lower for acetate and myristate than for palmitate, oleate, linoleate and arachidonate. It is possible that fatty acids within the cell may be able to act directly upon cellular components such as enzymes or gene expression to control metabolic processes such as glycolysis. The key enzyme at the beginning of the glycolytic pathway, hexokinase, is active in Hep2 cells (17.8 nmoles/min/mg protein) and such an inhibitory effect of saturated fatty acids upon hexokinase mRNA expression could cause direct inhibition of the glycolytic pathway~ resulting in the decreased glucose utilization rates seen in the present study. While this possible direct effect has not yet been studied in Hep2 cells, there is evidence in the recent literature of the regulatory role of unsaturated fatty acids upon enzymes of both glycolysis and lipogenesis, principally in the liver (16,17). In the liver, expression of mRNA for the glycolytic enzymes glucokinase (hexokinase type IV) and pyruvate kinase can be regulated by the polyunsaturated fatty acids eicosapentaenoate and docosahexaenoate (menhaden oil) and linoteate (olive oil) in the diet after very" short time periods (<3 hours) (16). Strikingly, the Hep2 cell has been previously shown by the author to contain an additional, unusual form of hexokinase with the kinetic properties of glucokinase (18). This activity (1.1 nmoles/min/mg protein) was shown to be sensitive to the ghicokinase-specific inhibitor mannoheptulose, concomitant with both decreased glucose utilization and decreased rates of proliferation when exposed to mannoheptulose in culture. This is of particular relevance to the aforementioned studies where unsaturated fatty acids have inhibitory effects on glucokinase mRNA expression and suggests that glucokinase may be one of the target enzymes affected by fatty acids in the present work. If the mRNA 605 Vol. 41, No. 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL expression of hexokinase and pyruvate kinase (1854 nmoles/min/mg protein) in Hep2 cells were inhibited by unsaturated fatty acids this could be a direct cause of inhibition of glucose utilization. Additionally, mRNA expression of glucose-6-phosphate dehydrogenase, a key enzyme of ribose-5-phosphate synthesis, at the branch point between glycolysis and the pentose phosphate pathway, is also regulated by unsaturated fatty acids in the liver (! 7,19). If this enzyme was also regulated in Hep2 cells (16.3nmoles/min/mg protein) by unsaturated fatty acids this would likely have a cumulative effect on inhibition of glucose utilization. Such effects on glucose utilization may thus have direct inhibitory effects upon the rate of cell proliferation as glycolysis is considered to be the most important source of ATP in tumor cells (20) in addition to the partial oxidation of glutamine (21,22). Indeed, the fatty acids found to be inhibitory to glucose utilization were inhibitory to cell proliferation while those which did not inhibit utilization did not inhibit proliferation. The rates of inhibition of proliferation were as follows: acetate, 0%, myristate, 8%, palmitate, 17%, oleate, 30%, linoleate, 73%, arachidonate, 70% (unpublished data). Inhibition by the fatty acids was not, however, attributable to direct cytotoxic effects as determined by trypan blue exclusion and lactate dehydrogenase leakage, viability being between 95.5 - 99.2% and enzyme leakage being between 1.3 - 4.0% of total cellular activity (unpublished data). In summary, Hep2 human larynx tumor cells incorporate fatty acids into intracellular components or oxidize them to C Q depending on the individual fatty acid. This partitioning of fatty acids is neither dependent on chain length nor degree ofunsaturation. The inhibitory effects of unsaturated fatty acids on glucose utilization by Hep2 cells is not linked to the individual rates of fatty acid oxidation, suggesting that a direct effect of the fatty acid upon cellular metabolism may exist. 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