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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.
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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
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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).
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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
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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
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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
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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
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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
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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
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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. It is proposed that the inhibition of gene expression of one or
several key enzymes of glucose metabolism may be involved in this process, such as
glucokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase. Since the fatty acids
inhibitory to glucose utilization were inhibitory to cell proliferation further study of the
actions of unsaturated fatty acids on both lipid and glucose metabolism, in tandem, may
clarify their potentially important regulatory and therapeutic role in the control of human
tumor cell proliferation.
ACKNOWLEDGEMENTS - A.C. was supported by CNPq during the course of this work
and the research was supported by FAPESP.
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