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1.
John A. Watson 《Lipids》1972,7(2):146-155
Hepatoma tissue culture (HTC) cells (derived from minimal deviation hepatoma 7288C) were cultivated in a complete medium containing
either glucose, fructose or glycerol as the primary carbon source. Growth was rapid on glucose and very low on fructose containing
media. Glycerol did not support any growth. Glucose-14C (U) and fructose-14C (U) are incorporated into the total lipid fraction of HTC cells. However the level of conversion of glucose into lipid is
much greater than fructose. Tritiated water is rapidly incorporated into the saponifiable and nonsaponifiable lipid fractions
of growing HTC cells. The level of incorporation is greater than that observed with glucose-14C (U) and the difference is constant over the experimental period studied. Lipoprotein poor serum (LPPS) isolated from a calf-fetal
calf serum mixture (1∶1) supported growth at a similar rate as the unfractionated serum combination (DS). However the total
lipid and total cholesterol content of the fractionated serum was one sixth and one fiftieth the level found in the whole
serum mixture, respectively. The level of incorporation of glucose-14C (U), acetate-14C (U) and tritiated water into the nonsaponifiable fraction of HTC cells grown on LPPS was 3 to 8-fold greater than that for
DS. However mevalonate-2-14C incorporation was stimulated only 1.3 to 1.7-fold. In general there was a much smaller response in the level of incorporation
of radioactive metabolites into the saponifiable lipids. From these studies and additional data, it was tentatively concluded
that HTC cells can respond to nutritional pertubations caused by changes in the exogenous lipid content. It was not determined
if the apparent responsiveness in the lipids of the nonsaponifiable fraction is due to “feedback control” or some other regulatory
mechanism.
One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971. 相似文献
2.
1-14C-Acetic, 1-14C-palmitic, or 1-14C-stearic acid was incubated with minimal deviation hepatoma 7288C cells grown in culture to assess: de novo fatty acid synthesis, oxidation, desaturation, and elongation of saturated fatty acids, as well as the ability of media fatty acids to serve as precursors of cellular glycerolipids. Distribution of radioactivity in the individual lipid classes and the various fatty acids of triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was determined. The radioactivity among the monoenoic acid isomers derived from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine was analyzed by reductive ozonolysis. Only small amounts of the labeled substrates were oxidized to carbon dioxide. Except for labeled stearic acid, which also was incorporated heavily into phosphatidyl inositol and phosphatidyl serine, most radioactivity was recovered in triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine. Synthesis of cholesterol and long chain fatty acids from labeled acetic acid demonstrated that these cells can perform de novo synthesis of fatty acids and cholesterol. Both labeled palmitic and stearic acids were desaturated to the corresponding delta9 monoenes, and palmitic and palmitoleic acids were elongated. The nexadecenoic acid fraction isolated from triglyceride, phosphatidyl choline, and phosphatidyl ethanolamine, when acetic or palmitic acid was the labeled substrate, showed that greater than 70 percent of the labeled acids were the delta9 isomer. Radioactivity of the octadecenoic acid fraction derived from labeled acetic or palmitic acids was nearly equally divided between the delta9 isomer, oleic acid, and the delta11 isomer, vaccenic acid. Desaturation of labeled stearic acid produced only oleic acid. These data demonstrate that the biosynthesis of vaccenic acid in these cultured neoplastic cells proceeds via the elongation of palmitoleic acid. The relatively high level of vaccenic acid synthesis in these cells suggests that the reported elevation of "oleic acid" in many neoplasms may result from increased concentration of vaccenic acid. 相似文献
3.
Minimal deviation hepatoma 7288 C cells were cultured on media containing 25% serum to the confluent stage. The growth media was replaced with serumfree media containing 1-14C-palmitate, and incubations were continued for 0.75, 1.5, 3, 6, 12, and 24 hr. The distribution of radioactivity among the major neutral lipids and phosphoglycerides was determined for cells and culture media. Radioactivity in individual fatty acids of cellular triglyceride, phosphatidylcholine, and phosphatidylethanolamine also was determined. After 24 hr, more than 95% of the administered radioactivity was recovered in neutral and phosphoglycerides, indicating that only a small amount of the fatty acid was oxidized. At any time period examined, over 80% of the incorporated radioactivity was found in triglyceride, phosphatidylcholine, and phosphatidylethanolamine. Incorporation of the label into cellular triglyceride and phosphatidylcholine plateaued at 12 hr, whereas incorporation of radioactivity into phosphatidylethanolamine still was increasing at 24 hr. In contrast, during the entire incubation period the relative distribution of14C among esterified lipid classes in the culture media remained constant. Elongation of palmitic acid to stearic acid and its subsequent desaturation to oleic acid suggests that these cells possess an active elongation and monoenoic desaturation system. Labeled glycerol ether diesters were not detected in the cells or culture media. Positional distribution of the14C label in the triglyceride and phosphatidylcholine suggests that minimal deviation hepatoma cells do not exhibit diglyceride selectivity in the biosynthesis of these two lipid classes. 相似文献
4.
Phosphatidylcholine. phosphatidylethanolamine, and triglycerides were isolated from minimal deviation hepatoma 7288C cells cultured as monolayers to confluency in roller flasks containing Swim's 77 medium supplemented with 5% fetal calf serum, plus 20%, 10%, or 5% bovine serum. Fatty acid distribution at each position of glycerol was determined for the 3 glycerolipid classes, and carbon number distributions of triglycerides and diglycerides derived from phosphatidylcholine and phosphatidylethanolamine were quantitated by high temperature gas liquid chromatography. Fatty acid composition was only marginally affected by the level of bovine serum in the culture medium. Percentage composition of fatty acids esterified at each position of the 3 glycerolipids was different, indicating a nonrandom distribution of acyl groups in triglycerides and the 2 diacyl phosphatides. The carbon number distribution of diglycerides derived from phosphatidylcholine and phosphatidylethanolamine was different, and neither carbon number distribution agreed with the calculated 1-random, 2-random diacyl distribution, thus indicating pairing of certain acids in the diglycerides derived from these phospholipd classes. The determined triglyceride carbon number distributions did not show complete agreement with those calculated, assuming a 1-random, 2-random, 3-random type of fatty acyl distribution, suggesting preferential pairing of some acids in this lipid class. The 1-, 2-diglycerides derived from phosphatidylcholine, phosphatidylethanolamine, and triglycerides differed, indicating either selectivity in utilization of diglyceride species in biosynthesis of these glycerolipids, or modification of glycerolipids after their initial synthesis. 相似文献
5.
Δ5 Desaturation of eicosa-8,11,14-trienoic acid to arachidonic acid was studied in rat liver microsomes. It was shown that
Δ5 desaturation of fatty acids in vitro requires the participation of a peripheral component of cytosolic origin. Desaturation
of 20∶3n−6 to 20∶4n−6 decreases in washed microsomes as they lose an adsorbed cytosolic fraction (CF), but the enzymatic activity
can be recovered as a function of CF concentration in the incubation medium. Albumin does not substitute for CF. Δ5 Desaturation
of 20∶3n−6 is inhibited by arachidonic acid by a product inhibition effect, but CF prevents retroinhibition of Δ5-desaturase
by 20∶4n−6. This ability of CF is eliminated by preincubation of CF with 20∶4n−6, but not with γ−18∶3n−6, the product of Δ6
desaturation of 18∶2n−6, thus indicating that CF impairs the retroinhibitory effect of arachidonic acid on Δ5-desaturase in
a specific manner. Δ6 Desaturation of linoleic acid to γ−18∶3n−6 is also activated by CF and retroinhibited by γ−18∶3n−6.
CF activity on Δ6 desaturation is retained after preincubation with 20∶4n−6, but it is lost after preincubation with γ−18∶3n−6.
Activation of Δ6-desaturase by CF is associated with the removal of the reaction product in a specific manner. Chromatography
of CF by Sephacryl S-200 separates two major subfractions which show different efficiency in reactivating Δ5- and Δ6-desaturase
activities in washed microsomes. Therefore, CF may contain subfractions that can prevent Δ5- and δ6-desaturase retroinhibition
by apparently binding their respective reaction products specifically. 相似文献
6.
R. R. Brenner 《Lipids》1969,4(6):621-623
Variable concentrations of [I14C] linoleic acid and [I14C] eicosa-8,11,14-trienoic acid were incubated with liver microsomes in a medium containing the necessary cofactors for fatty
acid desaturation. The conversion of linoleic into γ-linolenic acid and eicosatrienoic into arachidonic acid were mutually
inhibited and the inhibition depended on the concentration of the fatty acids incubated. 相似文献
7.
The effects of 2-hexadecynoic acid on the growth and lipid metabolism of cultured 7288 (HTC) cells have been evaluated. Growth
was inhibited by the acetylenic acid: the LD50 was 35–85 μM as determined by two methods at low and high cell densities. Reduced growth did not result from damaged plasma
membranes as determined by α-amino isobutyrate leakage. DNA synthesis was unaffected by the acetylenic acid and the effect
on RNA and protein synthesis appeared to be secondary to the effects on lipid metabolism. The 2-hexadecynoic acid inhibited
lipid metabolism of the HTC cells at least at two levels. Data from both mass studies and radioactive acetate distributions
in cellular and media lipids indicated that fatty acid elongation and acylation, especially triglyceride synthesis, were inhibited. 相似文献
8.
The inhibitory effects of the positional isomers oftrans-18∶1 acids on the desaturation of palmitic acid to palmitoleic (Δ9-desaturase), linoleic to γ-linolenic (Δ6-desaturase) and
eicosa-8,11,14-trienoic to arachidonic acid (Δ5-desaturase) were investigated. Thesetrans-18∶1 acids were found to be inhibitory for the microsomal Δ6-, Δ9- and Δ5-desaturases of rat liver. The position of the double
bond in thetrans-18∶1 acids seems to be important in determining the degree of inhibition. At inhibitor/substrate ratio of 3∶1, the Δ6-desaturase
was most strongly inhibited bytrans-Δ3,-Δ4,-Δ7 and-Δ15-18∶1 isomers, whereas the Δ9-desaturase was most strongly inhibited bytrans-Δ3,-Δ5,-Δ7,-Δ10,-Δ12,-Δ13 and-Δ16 isomers. At inhibitor/substrate ratio of 6∶1, the Δ5-desaturase was most strongly inhibited
by Δ3-, Δ9-, Δ13- and Δ15-isomers. When 18∶0 was added to the incubations of 16∶0, 18∶2 and 20∶3 at the same I/S ratios used
for thetrans-18∶1 acids, weak inhibition for Δ9-desaturase and no inhibition for Δ5-and Δ6-desaturases was observed. 相似文献
9.
The microsomal stearoyl-CoA desaturase system was examined in both the Morris hepatoma 7288CTC, maintained in the host Buffalo
strain rat, and the Morris hepatoma 7288C, maintained in tissue culture. In vitro examination shows the stearoyl-CoA desaturase
system to be similar in the 2 tissues. Both show extremely low overall stearoyl-CoA desaturase activity, having 4% and 8%
of normal liver values respectively. Examination of the electron transport system showed both tissues have decreased electron
transport components cytochrome b5 and cytochrome b5 reductase. Particularly noticeable were the extremely low levels of cytochrome b5 (2% compared with normal liver). Microsomes from both tissues showed a decreased ability to reduce an artificial electron
acceptor, cytochrome c. With the low levels of cytochrome b5 observed in these tissues, the low levels of overall desaturase activity may be caused by lack of terminal enzyme, lack of
sufficient cytochrome b5, or both. Analysis of the stearoyl-CoA desaturase system in cultured hepatoma cells suggests that these cells are similar
to the host-grown tumor in this respect and may be used as a model in further examinations of the stearoyl-CoA desaturase
system. 相似文献
10.
Fatty acids ofSterculia foetida were added to the medium used to maintain the Morris hepatoma 7288C in culture. The effect of this supplement on the lipid
composition was examined. Overall, monoene levels were decreased with 18∶1 levels reduced by 40%. Saturated fatty acid levels
were increased, with stearate (18∶0) levels 220% of control values. No effect occurred on the level of polyunsaturates (18∶2,
20∶4, 22∶5, 22∶6). These changes in fatty acid makeup were observed in both neutral and phospholipid fractions, and all lipid
classes were affected. Triglycerides were most affected with a 66% decrease in 18∶1. There appeared to be little specificity
of effect in the phospholipids with 18∶1 levels decreased 40–60% in all classes. All classes were therefore dependent on an
endogenous supply of 18∶1. Examination of the distribution of geometrical isomers of 18∶1 reveals that in all lipid classes,
except diphosphatidylglycerol (DPG), the ratio of Δ11 to Δ9 isomer decreased toward the isomeric distribution displayed by
total medium lipids. In DPG, although 18∶1 levels were lowered, the isomeric distribution increased. DPG, synthesized and
found in the mitochondria, may use a separate pool of 18∶1 during synthesis. Cyclopropene fatty acids (sterculic and malvalic)
were incorporated into both neutral and phospholipid fractions with preferential incorporation into triglycerides. Cyclopropene
fatty acids were not selectively incorporated into any phospholipid species. Sphingomyelin did not incorporate cyclopropene
fatty acids, indicating that a different class of acyltransferase is used in the formation of this phospholipid class. 相似文献
11.
The metabolism of [1-14C]eicosa-11,14-dienoic acid was investigated in human testes using whole tissue minces and microsomal preparations. Both types
of preparations catalyzed the desaturation of the labeled diene to eicosa-8,11,14-trienoic as well as eicosa-5,11,14-trienoic
acid. The reported results, therefore, indicate that human testicular tissue, as well as rat testicular tissue (reported previously),
is capable of utilizing eicosa-11,14-dienoic acid as a precursor of arachidonic acid. Since it is known that there is no Δ8 desaturase activity in rat liver and brain, these studies support the concept that there is a tissue variation in this enzymatic
pathway. 相似文献
12.
The major octadecenoate isomers, oleate (Δ9) and vaccenate (Δ11), were measured in the plasma phospholipids of rats bearing
hepatoma 7288CTC as the tumor developed. The percentage of vaccenate decreased from 45% of the octadeceoate fraction at day
zero to 25% by the 15th day. A significant decrease (45% to 35%) in the percentage of vaccenate occurred by the sixth day,
well in advance of detectable tumor growth. The percentage of vaccenate continued to decrease as a function of time until
day 15, after which it remained constant. Detection of alterations in plasma phospholipids at an early stage of tumor development
in rats suggests that experiments should be carried out to determine if the same effects occur in humans. 相似文献
13.
The relative capacity of Morris 7777 hepatomas and livers of tumor-bearing rats to take up and subsequently metabolize intravenously
injected radiolabeled free fatty acids was investigated. The objective was to determine differences in lipid metabolism which
may affect the lipid composition previously observed in this tumor. Both tissues demonstrated comparable selectivity in the
uptake of palmitate, linoleate and arachidonate from blood, although the hepatoma took up one-tenth as much free fatty acid
per g wet wt as liver. A much greater percentage of fatty acid taken up by the hepatoma was converted to aqueous soluble radioactivity,
perhaps the result of oxidation. In the hepatoma, palmitate was incorporated into phospholipid molecular species in a pattern
similar to that observed for diglyceride, which suggested that phospholipid synthesis occurred predominantly de novo. On the
other hand, in liver, a large percentage of palmitate was incorporated into polyunsaturated phospholipid molecular species
that were not present in the diglyceride pool, which suggested significant incorporation by the acylation of monoacyl phosphoglycerides.
These studies indicate that the specificity for the uptake of fatty acids was not different in the two tissues; however, the
subsequent metabolic processes are markedly different. 相似文献
14.
Uptake and metabolism of fatty acids by Soybean suspension cells 总被引:1,自引:0,他引:1
Soybean suspension cultures very rapidly take up C16 and C18 fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer
into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines.14C-Palmitic and14C-stearic acids follow this sequence but are not desaturated, wherease14C-oleic and14C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit
a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA;
both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine. 相似文献
15.
Eicosa-5,8,11,14-tetraynoic acid or arachidonic acid or no supplement (controls) was given orally to rats maintained on a
fat free diet and the fatty acid composition of total lipids of several organs determined. No changes were noted in the total
fatty acid concentrations of the organs examined in the various groups. A decrease in the amount of arachidonic acid, 22∶4ω6
and 22∶5ω6 (as percent of total fatty acids), and an increase in the amount of 20∶3ω6 and linoleate were observed in total
lipids of several organs. In the group receiving the arachidonate supplement, there was less linoleate and 20∶3ω6 and more
arachidonate than in the controls. Both eicosa-5,8,11,14-tetraynoic acid and arachidonate supplements resulted in a decrease
in 20∶3ω9 in most organs studied. Generally, the most marked changes were seen in liver but, of the other organs examined
(heart, kidney, testis, brain, and adrenals), only the adrenals failed to show any significant differences between the controls
and each of the two supplemented groups. Although the experimental conditions preclude conclusive interpretation of the changes
observed, it is suggested that eicosa-5,8,11,14-tetraynoic acid was effective in inhibiting the conversion of linoleate to
arachidonate and the conversion of arachidonate to 22∶4 and 22∶5. 相似文献
16.
The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures
and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a
significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained
unaltered. These microtubule-disrupting drugs also diminished Δ9-, Δ5-, and Δ6-desaturase capacities. The effects produced
by COL were dose (0–50 μM) and time (0–300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO).
Dihydrocytochalasin B (20μM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation
of [14C]FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane
proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation
of unsaturated FA biosynthesis.
The authors are members of the Carrera del Investigador Científico del Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Argentina. 相似文献
17.
18.
Agonist-stimulated phospholipases release arachidonate, but not 8,11,14-eicosatrienoate, from human endothelial cells. One
source of the arachidonic acid is deacylation of 1-alkyl-2-arachidonoyl-glycerophosphocholine, with subsequent conversion
of some of the resultant lysophospholipid to platelet-activating factor. This study has compared the distribution of incorporated
8,11,14-[14C]-eicosatrienoate in alkylacyl-GPC and diacyl-GPC with that of [14C]arachidonate synthesized endogenously by desaturation of the 8,11,14-[14C]eicosatrienoate. Cells were incubated for 24 or 48 hr with 8,11,14-[14C]eicosatrienoate, and the resultant mixture of14C-fatty acids in the cellular lipids was characterized by gas chromatography. The choline phospholipids were then separated,
hydrolyzed with phospholipase C and derivatized to diradylbenzoates. Gas chromatographic analysis indicated extensive incorporation
of [14C]eicosatrienoate, as well as [14C]arachidonate, into alkylacyl-GPC. Although the ratio of esterified [14C]arachidonate to [14C]eicosatrienoate was greater in alkylacyl-GPC than in diacyl-GPC, the enrichment with [14C]arachidonate was far less than the ratio of arachidonate/eicosatrienoate released from these cells. These results thus support
the hypothesis that the acyl specificity of polyunsaturated fatty acid release is provided by the agonist-stimulated phospholipase
A2 rather than the composition of the alkylacyl-GPC. 相似文献
19.
The lipid fatty acid pattern of normal liver, host liver, and Novikoff hepatoma was determined by gas liquid chromatography,
and Δ6-desaturase activity for linoleic acid was measured in the microsomal fractions. The results showed that, in Novikoff
hepatoma, there is a correlation between the low content of arachidonic acid and the low activity of Δ6-desaturase, a key
enzyme in the biosynthetic pathway of this acid. 相似文献
20.
The effects of fractions isolated from thermally oxidized corn oil or olive oil on the metabolic activity of heart endothelial
and muscle cells were studied. Rat heart cells in culture, exposed to thermally oxidized fat components, took up more exogenous
1-14C-palmitic acid and incorporated more of it into the cell triacylglycerol fraction than when the cells were treated with fresh
fats. Particularly with the heated corn oil compared to fresh corn oil, much less of the radioactivity from the labeled palmitic
acid was deposited in the phospholipid fraction. Also, with heated corn oil when the incubation period was extended beyond
12 hr, there was a decline in the radioactivity retained in the triacylglycerol fraction of the heart muscle cells. When the
fresh fats were compared for14C-radioactivity incorporation into the heart cells, the olive oil gave much higher values, indicating a distinct difference
in response to the proportion of fatty acids supplied.
Presented in part at the AOCS Meeting, Chicago, September 1976. 相似文献