Aortic lipogenesis during aerobic and hypoxic incubation

Atherosclerosis Elsevier Publishing

Company,

359

Amsterdam - Printed in The Netherlands

AORTIC

LIPOGENESIS

CHARLES

F. HOWARD,

Department Department

of Primate Nutrition, Oregon Regional Primate Research Center, Beaverton, and the of Biochemistry, University of Oregon Medical School, Portland, Oreg. (U.S.A.)

(Received

DURING

AEROBIC

AND HYPOXIC

INCUBATION

JR.

August 12th, 1971)

SUMMARY

Atherosclerotic

rabbit aortas incorporate more [2-i%]glucose

into lipids when

incubated under hypoxic than aerobic conditions. This increase results from greater radiosubstrate incorporation into the glycerol moiety of glycerides and phospholipids. Fatty acids are synthesized mainly by elongation; synthesis of all fatty acids is less during hypoxia. Though the total amount of [1-iJC]acetate incorporated into lipids is the same during both incubation conditions, there is a redistribution so that this substrate equilibrates more into phospholipid glycerol during hypoxia. Fatty acid synthesis from [1-i4C]acetate is primarily by elongation and decreases during hypoxic incubation.

Key words: [I-l%]Acetate - Aerobic Glucose - Lipogenesis

vs. hypoxic

-

Atherosclerotic

aorta

-

[2-W]-

INTRODUCTION

The concentration of oxygen available to aortic tissues affects the aortic metabolism and the development of atherosclerosis. In rabbits exposed in vivo to hypoxic conditions, atherosclerosisi has been exacerbated with metabolic changes regardless of the cholesterol concentration in the die@-6; increased atherosclerosis is accompanied by increased total lipid content, especially cholesterol and triglyceride@. The incorporation of [1-Wlacetate into total lipids increased in normal calf aorta incubated

This work was supported by the National Institutes of Health, U.S.P.H.S. grants No. AM-12601 (C.F.H.), No. HE-09744 (Dr. 0. W. Portman), and FR-00163 (Oregon Regional Primate Research Center). Publication No. 554 from the Oregon Regional Primate Research Center. Abbreviations: TLC = thin-layer chromatography: GLC = gas-liquid chromatography. Atherosclerosis,

1972, 15 : 359-369

360

C. F. HOWARD,

anaerobically

rather than aerobically,

but incorporation

from [U-i%]glucose

JR.

decreas-

ed’. The studies tions

occur

reported

here were undertaken

in atherosclerotic

aorta

incubated

to determine aerobically

what lipogenic and

hypoxically.

variaTwo

radiosubstrates, [2-Wlglucose and [l-Wlacetate, were used; the % is so located in these radiosubstrates that extensive analyses of lipids and lipid moieties could be made

and

MATERIALS

conclusions

drawn

about

metabolic

alterations

in aortic

metabolism*.

AND METHODS

and aorta preparation

Animals

Female fed Purina

New Zealand

rabbits

(Hilltop

chow pellets coated with a solution

Lab Animals,

Chatsworth,

Calif.) were

of 1.4 g/kg of corn oil (Mazola) and 0.8

g/kg of cholesterols (U.S.P., Nutritional Biochemicals Co.) for 3 to 4 months. After the rabbits had been killed by cervical dislocation, the aortas from the descending arch to the point of diaphragm penetration were removed and flushed with 0.9 y0 saline, and fat and adherent material were stripped away from the adventitial side. The aorta was everted

over a glass rod ‘and the ends were wrapped

with Parafilm

to allow access of

the medium only to the intimal side of the aortas. The aortas were placed in tubes containing ca. 2.5 ml Krebs-Ringer with 2.8 mM

[2-r4C]glucose

(5.4 ,uCi/pmole)

or 2.8 mM

salt medium

[1-i4C]acetate

(10.4 &i/

pmole) (New England Nuclear, Inc., Boston). All aortas were incubated at 39” for 3 hours, but the degree of oxygenation varied. Twelve of the aortas had oxygen 95%-carbon

dioxide 5 y0 bubbled

and continuously through a rubber stopper

agitate stopper

through

from the bottom

of the tube to oxygenate

the medium. For the other 12, the glass rod was passed and placed in a tube containing incubation medium. The

was sealed and wrapped

with Parafilm

to prevent

air from gaining

access to

the medium. The entire assembly was rolled during incubation to ensure adequate movement of the medium over the intimal surface. Since the 12 aortas were divided between the two radiosubstrates, the number of animals for each group was six. At the end of incubation, the aorta was cut away from the rod and examined for the extent of atherosclerosis; from 70 to lOOo/o of the intimal surface was covered with raised, white lesions. The intima (average weight

plus media were stripped

away from the adventitia

= 550 mg) and analyzed.

Lipid analyses Aliquots were radioassayed by liquid scintillation counting at all steps of analysis. Intima + media were homogenized in chloroform-methanol (2:1), passed through a G-25 Sephadex column to remove nonlipid contaminationlo, and separated on TLC into lipid classesii. Lipids were visualized with 2,3-dichlorofluorescein, the silica gel was scraped into pipets, and the lipids were eluted with appropriate solvents. So that radiosubstrate contamination could be ruled out, duplicate extractions were carried out on tissue with either [2-Wlglucose or [1-Wlacetate added to the chloroAtherosclerosiS,

1972, 15: 359-369

AORTIC

LIPOGENESIS

form-methanol

DURING

homogenate.

AEROBIC

AND

Radioactivity

HYPOXIC

INCUBATION

in the phospholipids

361 from [2-14C]glucose

would have been no more than 1.4 o/o and 2.6 o/o respectively; and from [ I-i%]acetate there was no substrate contamination in the triglycerides. Some of the lipids were dried under nitrogen, hydrolyzed in ethanolic KOH, and extracted; it was advantageous to include a small amount

of toluene to ensure complete

cholesterol esters. The petroleum sate contained nonsaponifiable acidified hydrolysate

contained

hydrolysis,

especially

of

ether (b.p.r. 4060”) extract of the alkaline hydrolycompounds, the petroleum ether extract of the fatty acids, and the aqueous portion contained

of glycerides or glycerophosphate separated on GLC (HI-EFF-2BP

glycerol

of phospholipids. Fatty acids were methylated and on Chromosorb W [AW], Applied Science Laborato-

ries, Inc.). The GLC effluent passed through

a splitter

to allow collection

of individual

fatty acids. These were decarboxylated 12 to determine the mechanism of fatty acid synthesis. The decarboxylation ratio of total fatty acid radioactivity/carboxyl radioactivity

is 1 if only elongation

occurs but is greater

(e.g., 8 for stearic acid) if de novo

synthesis

is a major process. Glycerophosphate from phospholipids was purified on and treated with acid phosphatase Dowex-1 acetate column chromatography13 (Calbiochem. Inc.). Each sample of glycerol from glycerophosphate or from triglyceride glycerol was diluted with carrier glycerol and passed through a mixed bed resin (Bio-Rad

Ag 501-X8

radioactivity metabolically

in carbon from

[D]). The purified 1 plus carbon

[2J*C]glucose,

glycerol

was degraded8

3 and in carbon

was degraded

8~4

to determine

> 95% of the activity was in carbon 1 so that decarboxylation ratios of fatty synthesized from [2-i*C]glucose can be compared directly to ratios obtained fatty

acids synthesized

from substrate

the

2. [i*C]Acetate, which arises to determine carbon location; acids from

[I-i*C]acetate.

RESULTS

Degree of hypoxia

To measure the degree of hypoxia attained during incubation, several control atherosclerotic aortas were incubated under conditions similar to those of the 12 sealed, atherosclerotic aortas but with a Radiometer electrode, model 27 (Copenhagen) inserted in a side arm to monitor oxygen consumption. With 2.8 mM glucose, oxygen consumption during the first 3 to 5 min averaged 0.84 ,umoles 02 consumed/h/100 mg wet weight; the rate decreased to 0.28 between 6 to 14 min and to 0.13 pmoles 0s between 14 to 20 min. Thereafter the rate of oxygen consumption was negligible, ca. 0.04 pmoles/h/lOO mg even though the pOz was ca. 95 mm Hg. The combined rates would have accounted for no more than 65 to 70 o/o consumption of the total oxygen originally present in the incubation medium. Similar results were found when no substrate was present. The aorta could be removed from the incubation medium after 20 min when oxygen consumption was minimal and, when placed in fresh medium, the pattern and extent of oxygen consumption was the same as previously. Under these conditions, the atherosclerotic aortic tissue was considered to be hypoxic for > 95 y. of the time of incubation since oxygen, though available, was not maximally consumed except during the first few minutes. Atherosclerosis,

1972,

15: 359-369

362 TABLE

C. F. HOWARD, JR. 1

INCORPORATION

OF

ATHEROSCLEROTIC

[2-14C]c~ucos~ AND

[l-I~CIACETATE INTO LIPIDS

OF

HYPOXIA

AND

AEROBIC

AORTA

[2-‘4ClGlucose [1-‘4ClAcetate (pmoles incorpovatedlmg wet weight/S h) a 23.5 + 6.0 52.4 f 9.4

Aerobic Hypoxic

15.6 * 5.3 15.7 + 2.9

& Mean & S.E.M. with 6 rabbits in each group.

Radiosubstrate incorporation The striking difference in incorporation apparent in Table 1 represents the more than doubling of [2-Xlglucose incorporation when aortas were incubated under hypoxic conditions (P = 0.03). This difference reflects variations in incorporation into lipid classes and, to a lesser extent, variations in the location of label within the lipid. Except for fatty acid synthesis, the mechanisms by which [I-14C]acetate was incorporated into lipids vary from those of [2-W]glucose so that comparisons for total label incorporation are not included between the two different substrates. The distribution of label in the different lipid classes is presented in Table 2; Pvalues are indicated for those lipid classes showing statistically significant changes. Changes in fatty acids were not significant; since the 1,3_diglyceride cochromatographed with cholesterol in this system, that band and the 1,2-diglyceride band were combined in these calculations and their statistical differences are not recorded. Hypoxia caused an increase in the percentage of [2-l%]glucose incorporated into triglycerides and monoglycerides with a concomitant decrease in the percentage incorporation into phospholipids. The opposite was true of [ 1-l%]acetate where aortas

TABLE

2

PERCENTAGE HYPOXICAND

DISTRIBUTION* OF 14C FROM [w4C]CLUCOSE AEROBIC ATHEROSCLEROTICAORTAb

[l-i4C]ACETATE

aerobic

hypoxic

P

aerobic

1.7 & 0.3 26.4 f 1.3 0.5 * 0.1

0.2 + 0.1 39.7 + 3.5 0.5 f 0.2

0.001 0.006

26.9 f 3.1 18.3 f 2.1 5.8 f 0.9

5.1 * 0.2 1.7 f 0.3 64.6 f 1.5

IN LIPIDS OF

[ I-l%]Acetate

[2-14ClGlucose

Cholesterol ester Triglyceride Fatty acid Cholesteroldiglyceride Monoglyceride Phospholipid

AND

3.1 f

4.1 f

0.5

11.2 f 4.2 45.4 + 4.0

0.05 0.002

8.8 + 1.6 7.6 & 1.6 3.4 * 0.9

0.7

2.9 & 0.9

3.7 + 0.4 41.2 & 1.6

6.2 f 1.3 71.2 & 5.2

8 Percentage of the total radioactivity recovered from TLC. b Values are the means & S.E.M. from each group of 6 ribbits. A thevosclerosis, 1972, 15: 359-369

hypoxic

P

< 0.001 0.03

< 0.001

AORTIC LIPOGENESIS DURING AEROBIC AND HYPOXIC INCUBATION

incubated

under hypoxic

conditions

incorporated

363

a lesser percentage

in triglycerides

and more in phospholipids. Under both incubation conditions, a significant amount of the radioacetate incorporated was in cholesterol esters but was greater when incubated aerobically. Since the amount

of [1-14Clacetate

same under both incubation cate changes in the amounts

incorporated

into the total

conditions, the percentage of substrate incorporation.

lipids was the

changes of Table 2 also indiHowever, the [2-14Clglucose

incorporated increased with hypoxia so that even those lipids showing a percentage decrease in Table 2, e.g., phospholipid, actually had an increased amount of radiosubstrate

incorporation.

This is seen in Table 3 where the amounts

of the two radio-

substrates incorporated into different moieties of lipids are shown; values product of mean radiosubstrate incorporation (Table 1) and mean percent lipid

(Table

(METHODS).

metabolites

are the of each

determined by hydrolysis of the lipids* 2) with the 14C-distribution No corrections are made for the dilution of radiosubstrate by endogenous but corrections are applied for the loss of 14C during the passage of each

radiosubstrate through metabolic pathways. The amount of [2-i%]glucose in fatty acids is doubled since i4C in the trioses leading to acetyl coenzyme A arises from equilibration of [2-14Cldihydroxyacetone phosphate with unlabeled glyceraldehyde phosphate (Fig. 1) after cleavage of [2-14Clglucose. The values for [1-14C]acetate appearing in glycerol are also doubled since the passage of radioacetate through the citric acid cycle to trioses results in the loss of half of the 14C. In all lipids examined, the incorporation of radiosubstrate moiety was less under hypoxic than under aerobic incubation. fatty

acids are accounted

for in the three lipids reported

into the fatty acid About 90% of the

in Table 3; the remaining

approximately 10% showed similar decreases with hypoxia. The decline in radiosubstrate incorporation was cu. 1.5fold for fatty acids synthesized from [2-i4C]TABLE

3

AMOUNTSOF [%14C]GLUCOSE OF ATHEROSCLEROTIC

AND

AORTAS

[I-14c]ACETATE

UNDER

HYPOXIC

INCORPORATED AND

AEROBIC

INTO

FATTY

ACIDS

AND

GLYCEROL

CONDITIONS

JAcetate [l-l% (pmoles radiosubstrate incovporatedlvng wet wt.13 h)

[2-14C]Glucose

aerobic Triglyceride Fatty acid Glycerol Phospholipid Fatty acid Glycerol Cholesterol ester Fatty acid Cholesterola * This fraction,

0.4 6.0

1::: 0.8 0.02 when measurable,

hypoxic

aerobic

0.2 20.7

2.0 1.8

0.8 0.7

2.::;:

4.7 3.4

2.8 16.5

0.2 -

4.1 0.15

was not identified specifically

ILypoxic

1.3 0.08 as cholesterol.

A thevosclevosis, 1972, 15: 359-369

364

C. F. HOWARD, JR. CHO H*+OH H$zH

[2%3

GLUCOSE ??

HiOH

-

6~~0~ 1

t t

?? CHO

.CHIOH &0

??

HtOH s 6 H ,O,J m-s----d II

b~,Op

?? CH20H -H&OH ---. -) _---- + kH,OH 1 0 ‘I H,t-0-?-CH,-R

t 0 Rt_CH,$.O+H

1’ i II

?? COOH

’ e. H,C-0-C-CH,-•CH,-CH,-R”

+=o

f

??

: :

I

‘\\ ‘\ *

?? yOOH yHo

/

:

0

‘\\

,/

CH,-$OH

‘\ [I-‘4C]

/ ,.*,,/

TRIGLYCERIDE,

ACETATE

2 kOOH

Fig. 1. Pathways showing (C* -+) and [I-lK]acetate

incorporation of radioactive (Co - - - -+ ).

carbon

into lipids from

[L-l‘X]glucose

glucose and cu. 2.2-fold for those arising from [1-r%]acetate.

As might be expected

from the fewer number of enzymic steps to be traversed,

[1-r%]acetate

showed

greater incorporation into fatty acids than [2-r%]glucose. However, even with the potential for greater dilution of the [2-r%]glucose metabolites passing through more enzymic steps, this radiosubstrate was 20 to 50 o/oas effective in fatty acid synthesis as [1-r4C]acetate. The incorporation of both substrates into the glycerol moiety was greater under hypoxic than aerobic conditions, except for the lesser amounts of [1-r%]acetate incorporated into triglyceride glycerol. The increase of 1% from [2-14Clglucose appearing in triglyceride glycerol was 3.4-fold, that in phospholipid glycerophosphate was only 1.6-fold. That label in I%-glycerol from [2-rK]glucose reflects net synthesis but 1% from [1-r%]acetate represents radiosubstrate equilibration with endogenous metabolites and passage through the citric acid cycle and glycolysis (Fig. 1). Substantial amounts of label from radioacetate appeared in phospholipid glycerophosphate. Atherosclerosis,

1972, 1.5:359-369

AORTIC

LIPOGENESIS

TABLE

4

DURING

AEROBIC

DISTRIBUTIONOF 14c IN THE CARBONS

AND

HYPOXIC

OF TRIGLYCERIDE

365

INCUBATION

GLYCEROL

AND

PHOSPHOLIPID

GLYCERO-

PHOSPHATE=

Carbon

7-14CjAcetate

j2-~4cjG1~~0~~

[

aerobic

hypoxic

Pb

aerobic

hypoxic

Pb

< 0.001

Triglyceride

1+3 2

5.5 94.5

2.2 97.8

< 0.001

98.2 1.8

85 15

Phospholipid

1+3 2

6.8 93.2

2.1 97.9

< 0.001

95.6 4.4

c c

& Values are the means of 6 determinations. b Statistical significance between radiocarbon tions. c Insufficient radioactivity for analysis.

distribution

following aerobic vs. hypoxic incuba-

The activity in cholesterol ester resided primarily in the fatty acids with less in the alkaline petroleum ether extract, presumably cholesterol or similar sterols. The possibility of activity at cholesterol ester being due to squalene was ruled out by hydrolysis and rechromatography; no radioactivity then migrated to the cholesterol ester spot, which in this system cochromatographs with squalene, nor was any radioactivity evident at a squalene spot when cholesterol ester was run in a less polar TLC system. The distribution of 1% in the glycerol moiety from both substrates is presented in Table 4. Some randomization

of r4C from [2-r%]glucose

is apparent in the two

different lipids; this decreases with hypoxia. For [ 1-r%]acetate, only minimal randomization of label occurred during aerobic incubation but more was evident during hypoxia; the latter changes are in keeping with the increased [I-r%]acetate tion and incorporation into glycerol with hypoxia.

equilibra-

Fatty acid synthesis from [2-r‘K]glucose was primarily by de novo synthesis for C 14:0 and C 16:O (Table 5) and by elongation for C 18:0 and C 18:l; longer fatty acids (not shown) also had radiosubstrate incorporated by elongation. A higher percentage TABLE

5

DECARBOXYLATION

Fatty

14:o 16:O 18:0 18:l

acidb

RATIOS

OF PHOSPHOLIPID

FATTY

ACIDSa

[l-14C]Acetate

[2J4C]Glucose aerobic

hypoxic

aerobic

hypoxic

6.7 7.5 1.0 1.1

C C 1.0 1.0

5.3 6.2 1.2 1.2

4.4 4.8 1.0 1.0

& Values are the mean of 3 to 4 decarboxylations. b Length of carbon chain:number of double bonds. c Insufficient radioactivity. Atherosclerosis,

1972, 15: 359-369

366

C.

of C 14:0 and C16:O arose by elongation fatty

acids

accounted

were again for only

synthesized

from [1-i%]acetate exclusively

whereas the longer chain De novo synthesis

by elongation.

10 to 15% of the radiosubstrates

F. HOWARD, JR.

incorporated

into fatty

acids.

DISCUSSIOIU

In vivo hypoxia

increases

atherosclerosisi-6,

with marked

changes

in the struc-

ture and metabolism of the aorta. There is some disagreement whether consumption of oxygen in atherosclerotic aorta is greateris-17, remains unchanged’s, or is less than in normal aortic tissueis. MAIER~~ has pointed out that the amount of oxygen consumption is affected by the duration of cholesterol feeding; additional variables among workers include the substrate utilized, tissue section studied, the method of tissue preparation oxygen

(especially

consumption.

temperature Very little

effect&ss),

is known

and

about

the method

for measuring

the effects of hypoxia

on aortic

lipogenesis7, especially with the development of atherosclerosis. It has been shown previously8 that lipogenesis from both [2-%]glucose and [I-i%]acetate is increased by atherosclerosis in aortas incubated aerobically. The work reported directly with lipogenic differences in hypoxic and aerobic atherosclerotic

here deals aorta by

using whole aorta preparations with only the intimal side exposed to the incubation medium. Hypoxia was attained by allowing the aorta to consume that oxygen still present in the incubation

medium

until

there was only minimal

uptake.

As noted here and

elsewheress, this effect was rapid so that moderate to severe hypoxia was present for 95-98 y. of the incubation time. The fact that aortic hypoxia would exist even though some oxygen

was still present

in the incubation

medium

reflects the inability

of the

aorta to take up or utilize the oxygen. It also emphasizes the need, when studying expose the incubation medium to aeration to ensure aorta ilz vitro, to continuously that the aorta is in a sustained aerobic milieu. The amounts of radiosubstrate incorporated

and the specific distribution

the moieties of lipids were affected by hypoxia. The greatest in lipids was from [2-iX]glucose with no increase apparent

into

increase of radioactivity from [1-i4C]acetate; the

increase from radioglucose is in keeping with the in vivo observations of increased lipid accumulation observed in atherosclerosis induced by hypoxias+s. The incorporation of the two radiosubstrates into the lipids and lipid moieties is via different metabolic pathways8 and the increase of radioglucose represents net accumulation whereas that from radioacetate reflects mainly equilibration of 1% with endogenous metabolites (with the exception of fatty acid synthesis). The observations reported here are unlike those of KRESSE et ~1.7 who found accumulation of [l-i4C]acetate radioactivity but not of [U-i%]glucose in lipids of normal calf aorta when the effects of oxygen were studied. The major increase of [2-r%]glucose in lipids of aortas incubated hypoxically was in the glycerol moiety of phospholipids and glycerides (Table 3). Aorta has active Atherosclerosis,

1972, 15: 359-369

AORTIC LIPOGENESIS

DURING AEROBIC AND HYPOXIC

INCUBATION

glycolysis22124-27 with 75 to 85% of glucose metabolized increases

the amount

anaerobic formation

metabolized

acetate

incorporation

radiosubstrate

accumulation

would

represent

with endogenous

to lactic acid; anaerobiosis

to lactic acid 24-26, but only by about 30 %. With

conditions there would be some increase and recycling of metabolites to glycerol.

crease in glycerophosphate

367

in trioses available This could account

from [2-i%]glucose; greater

metabolites

equilibration that

for glycerol for a net in-

the increased

[l-r%]-

and recycling

of that

are eventually

incorporated

into

glycerophosphate. Increased fatty

glycerophosphate

acids. However,

fatty acids decreased be due to an actual radioactive

acetyl

the total

would provide amount

of either

more substrate

for esterification

radiosubstrate

incorporated

of into

1.52.2-fold when aortas were incubated hypoxically; this could reduction in fatty acid biosynthesis or to greater dilution of

units

by endogenous,

nonradioactive

acetyl

units.

Since the 1%

glycerol from [2-i%]glucose increased in triglycerides and phospholipids, there would be increased esterification of fatty acids. The exact origin of these fatty acids would be in doubt; no exogenous fatty acid substrate was supplied and synthesis, as reflected by radioactivity

incorporation,

was diminished.

Active lipases, esterases,

and phospho-

lipases present in aortazs-31 do change with aging and atherosclerosis and could provide varying amounts of fatty acid substrate for esterification. The resultant lipid patterns found here could be due either to the increased hydrolysis of lipids under hypoxic conditions to make more fatty acids available for esterification or to a sufficiently high concentration of fatty acids hydrolyzed from endogenous lipids but whose hydrolysis was unaffected by the degree of oxygenation. Further data are needed before the exact control mechanisms effecting these changes in lipogenesis with hypoxia are known. Certainly the controls of glycolysis and gluconeogenesis in other tissues32 are not wholly applicable here. The preponderance of glycolysis with little tricarboxylic acid cycle and a minimal Pasteur effect24 accounts only partly for the greater incorporation of the glycerophosphate moiety into lipids. The well-studied controls of de lzovo fatty acid synthesis33 are not entirely applicable here either since 85 to 90% of the aortic fatty acids arise by elongation; controls in this case may lie in the reduced production of activated acetyl units from mitochondria operating under hypoxic conditions to yield less precursor for fatty acid synthesis. Esterification of endogenously produced fatty acids could still continue, or even be greater during hypoxia, if the ATP generated by glycolysis in the cytoplasm was available to activate fatty acids before esterification by the endoplasmic reticulum. Additional in atherosclerotic

work is needed before the effects of hypoxia aortas can be understood.

on control mechanisms

ACKNOWLEDGEMENTS

I thank assistance.

Miss Lynne

Bonnett

and Mrs. JoAnn

Wolff for their

Atherosclerosis,

fine technical

1972, 15: 359-369

C. F. HOWARD, JR.

368 REFERENCES

B~~CHNER, F. AND U. LUFT, Hypoxlmische Veranderungen des Zentralnervensystems im Experiment, Be&. Path. Anat. A&. Pathol., 1936, 96: 549. 2 M~ASNIKOV, A. L., Influence of some factors on development of experimental cholesterol atherosclerosis, Circulation, 1958, 27: 99. 3 FILLIOS, L. C. AND G. V. MANN, The importance of sex in the variability of the cholesteremic response of rabbits fed cholesterol, Circ. Res., 1956, 4: 406. 4 KJELDSEN, K., J. WANSTRUP AND P. ASTRUP, Enhancing influence of arterial hypoxia on the development of atheromatosis in cholesterol-fed rabbits, J. Atheroscler. Res., 1968, 8: 835. 5 HELIN, P. AND I. LORENZEN, Arteriosclerosis in rabbit aorta induced by systemic hypoxia, Arzgiology, 1969, 20: 1. 6 HELIN, P., I. LORENZEN, C. GARBARSCHAND M. E. MATTHIESSEN, Arteriosclerosis and hypoxia, Part 2 (Biochemical changes in mucopolysaccharides and collagen of rabbit aorta induced by systemic hypoxia), J. Atherosclcr. Res., 1969, 9: 295. 7 KRESSE, H., I. FILIPOVIC AND E. BUDDECKE, Gesteigerte i*C-Inkorporation in die Triacylglycerine (Triglyceride) des Arteriengewebes bei Sauerstoffmangel, Ho+@-Seyler’s 2. Physiol. Chem., 1969, 350: 1611. 8 HOWARD, JR., C. F., Lipogenesis from [2-r%]glucose and [I-i%]acetate in aorta, J. Lipid Res., 1971, 12: 725. 9 FILLIOS, L. C., S. B. ANDRUS AND C. NAITO, Coronary lipid deposition during chronic anemia or high altitude exposure, J. Appl. Physiol., 1961, 16: 103. 10 WUTHIER, R. E., Purification of lipids from nonlipid contaminants on Sephadex bead columns, J. Lipid lies., 1966, 7: 558. 11 SKIPSKI, V. P., A. F. SMOLOWE, R. C. SULLIVAN AND M. BARCLAY, Separation of lipid classes by thin-layer chromatography, Biochim. Biophys. Acta, 1965, 106: 386. 12 PHARES, E. F., Degradation of labeled propionic and acetic acids, Arch. Biochem. Biophys., 1951, 33: 173. 13 LEWIS, N. AND P. W. MAJERUS, Lipid metabolism in human platelets, Part 2 (De noao phospholipid synthesis and the effect of thrombin on the pattern of synthesis), J. Cl&. Invest., 1969,48: 2114. 14 D’ A~AMo, JR., A. F. AND A. P. D’ADAMO, Acetyl transport mechanisms in the nervous system. The oxoglutarate shunt and fatty acid synthesis in the developing rat brain, J. Neurochem., 1968, 15: 315. 15 WHEREAT, A. F., Oxygen consumption of normal and atherosclerotic intima, Circ. lies., 1961, 9: 571. 16 FISHER, E. R. AND J. H. GELLER, Effect of cholesterol atherosclerosis, hypertension and cortisone on aortic oxygen consumption in rabbit, Circ. Res., 1960, 8: 820. 17 MANDEL, P., G. POIREL AND N. SIMARD-DUQUESNE, Oxygen uptake of normal and atherosclerotic rabbit aortae in mediums of normal and hyperlipaemic sera and plasmas, J. Atheroscler. Res., 1966, 6: 463. 13 CHATTOPADHYAY, D. P., Influence of experimental atherosclerosis in rabbits on the rate of respiration and glycolysis by aortic tissue slices, Arzn. Biochem. Exp. Med., 1962, 22: 77. 19 MUNRO, A. F., B. M. RIFKIND, H. J. LIEBESCHEUTZ, R. S. F. CAMPBELL AND B. R. HOWARD, The effect of cholesterol feeding on the oxygen consumption of aortic tissue from the cockerel and the rat, J. Atheroscler. Res., 1961, 1: 296. 20 MAIER, N., Metabolism of arterial tissue and its relation to atherosclerosis, Ann. N. Y. Acad. Sci., 1968, 149: 655. 21 NAKATANI, M. ,T. SASAKI, T. MIYAZAKI AND M. NAKAMURA, Synthesis of phospholipids in arterial walls, Part 1 (Incorporation of ssP into phospholipids of aortas and coronary arteries of various animals), J. Atheroscler. Res., 1967, 7: 747. 22 SCOTT, R. F., E. S. MORRISON AND M. KROMS, Effect of cold shock on respiration and glycolysis in swine arterial tissue, Amer. J. Physiol., 1970, 219: 1363. 23 SIMARD-DUQUESNE, N. AND C. ALLARD, Evidence of an intimal metabolic barrier limiting the oxygen uptake of rat aorta, J. Atheroscler. Res., 1967, 7: 245. KIRK, J. E., P. G. EFFERS~E AND S. P. CHIANG, The rate of respiration and glycolysis by human and dorr aortic tissue, 1. Gerontol., 1954, 9: 10. ZEMPL~NYI, T., Enzymes of the arterial wall, J. Atheroscler. Res., 1962, 2: 2. SAVINO. E. A., H. C. MOGUILEVSKY, I. SZIJAN, L. GERSCHENSON AND J. DEPAOLI, Glucose metabolism in aorta of normal and gonadectomized rats, Acta Physiol. Lat.-Amer., 1965, 15: 403. JENSEN, J., An in vitro method for the study of cholesterol uptake at the endothelial cell surface of the rabbit aorta, Biochim. Biophys. Acta, 1967, 135: 532. 1

Atherosclerosis,

1972, 15: 359-369

AORTIC

LIPOGENESIS

DURING

AEROBIC

AND

HYPOXIC

INCUBATION

369

28 ADAMS, C. W. M., 0. B. BAYLISS, Y. H. ABDULLA, F. R. MAHLER AND M. A. ROOT, Lipase, esterase and triglyceride in the ageing human aorta, J. Atheroscler. Res., 1969, 9: 87. 29 PORTMAN, 0. W., Arterial composition and metabolism. Esterified fatty acids and cholesterol, Advan. Lipid Res., 1970, 8: 41. 30 PATELSKI, J., D. E. BOWYER, A. N. HOWARD AND G. .4. GRESHAM, Changes in phospholipase .4, lipase and cholesterol esterase activity in the aorta in experimental atherosclerosis in the rabbit and rat, J. Atheroscler. Res., 1968, 8: 221. 31 EISENBERG, S., Y. STEIN AND 0. STEIN, Phospholipases in arterial tissue, Part 4 (The role of phosphatide acyl hydrolase, lysophosphatide acyl hydrolase, and sphingomyelin choline phosphohydrolase in the regulation of phospholipid composition in the normal human aorta with age), J. Clilz. Invest., 1969, 48: 2320. 32 NEWSHOLME, E. A. AND W. GEVERS, Control of glycolysis and gluconeogenesis in liver and kidney cortex, Vitam. Harm. (New York). 1967, 25: 1. 33 VAGELOS, P. R., Lipid metabolism, Anx. Rev. Bzochem., 1964, 33: 139.

Atherosclerosis,

1972,

15: 359-369