Expression of hepatic microsomal cholesterol 7α-hydroxylase activity in lean and obese Zucker rats

Vol. 150, No. 2, 1988 January 29, 1988

Expression

of Hepatic Microsomal Cholesterol Activity in Lean and Obese Zucker Pauline

Department Northeastern

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 853-858

BIOCHEMICAL

November

10,

M. Tang, and John

7a-Hydroxylase Rats

Judith A. Finkelstein Y. L. Chiang”

of Biochemistry and Molecular Ohio Universities College Rootstown, OH 44212

Pathology of Medicine

1987

The activity of hepatic microsomal cholesterol 7o-hydroxylase was studied in genetically obese and lean Zucker rats. The liver microsomal cholesterol 7o-hydroxylase activity in fatty Zucker rats (fa/fa) is about 50% to 70% lower than that of the -lean (Fa/-) rats of the same sex, when animals were sacrificed at the middle of the dark cycle. When rats were sacrificed at the middle of the light cycle, cholesterol 7e-hydroxylase activity was the same as in the dark cycle in obese rats of both sexes, but was 65% lower in lean rats. However, cholesterol 7ol-hydroxylase activity was stimulated by the treatment with cholestyramine in both obese and lean rats. Our results suggested that the diurnal regulation of cholesterol 7o-hydroxylase activity is lost in obese rats but was present under cholestyramine treatment in the genetically obese strain of rats. 0 1988 Academic Press, Inc.

Microsomal major

pathway

(1).

This

cholesterol for

enzyme

oxygenase

and

mones , cytosolic

is

lo-hydroxylase

the

conversion

activity

is

regulated factors,

varies

Diurnal

rhythm

of cholesterol

(2,3).

Optimal

minimal nism

activity of

this

activity

was during

acids

regulation

is

should

observed

largely

enzyme

bile

The

species, activity

during

middle

and

(2).

on animal

the

to

compounds

7o-hydroxylase

occurs

*To whom correspondence Abbreviations sulfonate);

depending

rate-limiting acids

in

the

by a cytochrome-P-450-containing

by various bile

the

cholesterol

catalyzed

and

7cr-hydroxylase

of

is

the

of light

unknown.

factors

of

diet,

sex,

cycle.

of

and

Cholestyramine,

age

(2).

documented

dark

However,

hor-

cholesterol

has been well middle

liver mono-

including

activity

in the

cycle

and

the mechaa bile

acid

be addressed.

: CHAPS, (3-[(3-cholamidopropyl)dimethylanrmonio]-l-propaneHMG-CoA, 3-hydroxy-3-methylglutaryl-CoA

853

ooO6-291x/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any fom reserved.

Vol. 150, No. 2, 1988

sequestrant,

BIOCHEMICAL

is known to stimulate

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

cholesterol

sumably, by removal of the feedback inhibitor, down regulation following

of bile

the addition

Genetically

acid synthesis

of bile

model to study hypercholesterolemia

olemia, hyperphagia,

in rat hepatocyte

(fa/fa)

pre-

In contrast,

no

monolayer cultures

have been established as an animal

and obesity.

hyperglyceridemia,

The obesity

obesity,

is caused by a

by hypercholester-

and hyperinsulinemia

by a hormonal defect,

tion is enhanced and glucagon secretion

of cholesterol

acids.

The obese rat is characterized

The obese state is characterized

olemia could result

bile

activity,

acids was observed (4,5).

obese Zucker rats

recessive mutant gene (6).

7ci-hydroxylase

in which insulin

impaired (10,ll).

(7-9). secre-

The hypercholester-

from the increased synthesis and/or decreased degradation

in the liver.

pression of cholesterol

We report

here the diurnal

7cr-hydroxylase activity

Materials

regulation

and ex-

in obese and lean Zucker rats.

and Methods

12-week old obese (fa/fa) and lean (Fa/-) Zucker rats were obtained from a colony maintained at the Northeastern Ohio Universities College of Medicine. They were paired in plastic rat cages and maintained on a 12 hrs. dark-light cycle of either 2 a.m. to 2 p.m. light (normal) or 2 p.m. to 2 a.m. light (reversed) and had free access to regular Purina rat chow and water for 3 weeks before use. For the induction experiments, paired rats were fed 3% cholestyramine mixed in ground Purina rat diet for three weeks. All rats were sacrificed at 8 a.m., the middle of either the light or dark cycle. Four pairs of matched obese and lean rats were used for each experiment. Student's t-test was used to analyze significance. Rats were sacrificed by cervical dislocation. Livers were removed and minced in 0.05M Tris-acetate buffer, pH 7.5, containing 1mMEDTA, 1.15% KCl, and 50mMNaF. Microsomes were prepared from individual liver according to routine methods (12). Microsomal protein was determined by the‘method of Lowry (13). Cytochrome P-450 concentrations were determined according to &aura and Sato (14). Microsomal cholesterol 7a-hydroxylase activity was assayed by TLC-GC method developed recently (15). About one to two mg microsomes were incubated 1mM in O.lM potassium phosphate buffer, pH 7.4, containing 5mMdithiothreitol, The EDTA. Thin layer chromatography was double-developed in diethyl ether. 7$- and 7a-hydroxycholesterol were 0.83, 0.54 and RF values for cholesterol, 0.39, respectively. The area corresponding to 7a-hydroxycholesterol was scraped from the plates and extracted three times with diethyl ether. 7ahydroxycholesterol was derivatized with TRI-SIL-TBT and further separated by as an gas chromatography with a SP 2250 column. Using methyl-hyodeoxycholate

BIOCHEMICAL

Vol. 150, No. 2, 1988

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

internal standard, the relative values for 7a-hydroxycholesterol and cholesMicrosomes treated with terol were 0.51 and 0.61, respectively, at 235OC. absolute ethanol before adding the NADPH regenerating system were used as the background to substrate the nonenzymatically oxidized products.

Results Table

I

microsomes

shows

prepared

specific

activity

Activity

in

mates, ties protein

the from in

female

only

content

7o-hydroxylase

cholesterol

rats

male

rats

sex

in

was higher

was half

of

was less

than

one-third

both

specific

activity

lean in

at the middle

rats

differences

observed

in rats.

Although

Phenotype

Male

Obese Lean

Female

Obese Lean

(fa/fa) (Fa/-)

in

dark

cycle.

lean that

total

than

in obese

litter in

and total

in lean

Specific (pmol/min/mg

(Fa/-)

of

the

lean

enzyme liver

microsomal

activity

Total activity (pmol/min)

22.5

2 2.9"

3803

46.1

? 2.4

5532

24.3

+ 5.0"

3888

86.8

2 4.9

10,676

Paired obese and lean rats were fed a normal diet and maintained under reversed dark-light cycle (2 a.m. to 2 p.m. dark). Rats were sacrificed at 8 a.m., the middle of the dark cycle. Two mg of protein from each microsome were incubated in 0.1 M potassium phosphate buffer, pH 7.4, (containing 5 mM dithiothreitol, 1 mM EDTA, 2 units of glucose-6;phosphate dehydrogenase, 16.7 mM glucose-6-phosphate, 2 mM NADP , 0.015% CHAPS, and 0.1 mH cholesterol) for 20 minutes at 37OC in a final assay volume of 1 ml. Total activity was calculated by multiplying the specific activity by total microsomal protein per liver. Values *p
are (two

means tailed

? S.E., t-test)

n = 4. obese

855

vs.

lean

of

the

same

sex.

litter activi-

cholesterol

ACTIVITY

activity protein)

The

microsomal

rats.

7cr-hydroxylase

liver

mates.

I

CHOLESTEROL 7~HYDROXYLASE IN ZUCKSR RATS

(fa/fa)

in

liver

Cholesterol Sex

that

total

TABLE

activity

of the

rats,

obese

was higher

MICROSOMAL

7a-hydroxylase

sacrificed

obese

obese

Significant were

typical

and Discussion

Vol. 150, No. 2, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

II

INDUCTION OF CHOLESTEROL CHOLESTYRAMINE-FEEDING

7~HYDROXYLASE IN ZUCRER

ACTIVITY RATS

Cholesterol 7or-hydroxylase (pmol/min/mg protein) Male

Phenotype

A.

Dark

Female

cholestyramine diet

regular diet

rats cholestyramine diet

cycle

Obese Lean B.

activity

rats

regular diet

BY

Light

22.5 46.1

T 2.9 + 2.4

14.8 16.3

? 0.7 k 2.6

159 201.2

? 24 ?r 9.4

24.3 88.4

2 5 k 4.9

173.3 197.9

* 26.6 t 15.3

34.2 30.5

T 4.6 k 2.9

78.5 160.5

2 20.5 ?r 22

cycle

Obese Lean

66 -+ 30.1 94.7 t 12.4

Paired obese and lean rats were fed either a regular diet or diet supplemented with 3% cholestyramine and maintained under regular (2 a.m. to 2 p.m. light) Rats were sacrior reversed light (2 a.m. to 2 p.m. dark) cycle for 15 days. ficed at 8 a.m. A. Dark cycle refers to the group under reversed light cycle. Assay condiB. Light cycle refers to the group under regular light cycle. tions were the same as described under Table I and "Method" sections.

terol

are

Table

II

of either middle

of

was about

i S.E.,

shows

the

at

of both

the

activity dark

2- to

cholestyramine

in

the

cycle,

dark of

the

those

middle

lean

and induction

and obese

microsomal lean

rats

When lean

light

cycle

and sacrificed

cholesterol

activity

7a-hydroxylase on a normal

In

cycle.

choles-

rats.

maintained

light

of microsomal

Zucker

on a reversed

of the

7a-hydroxylase

contrast,

was absent

at

activity

light

cycle

diurnal

in genetically

Zucker

and

variation obese

Zucker

sexes. treatment

in

cycle,

4-fold

variation

maintained

Cholestyramine lase

diurnal

sex were

cholesterol

rats

n = 4.

activity

2.Gfold

sacrificed of

means

7a-hydroxylase

rats the

Values

liver the

in

significantly

stimulation

lean treatment

of

microsomes

was

lean

of activity The

rats.

increased

stimulation

also

observed 856

cholesterol

and obese

rats

was about

7-fold

of microsomal in

lean

and

7cr-hydroxy-

of both in

obese

enzyme obese

sexes.

In

rats

and

activity

by

rats

of

both

Vol. 150, No. 2, 1988

sexes

when

ation

of

fatty

Zucker

BIOCHEMICAL

sacrificed

in

cholesterol

the

after

treatment

did

microsomal

cholesterol

not

the

change

synthesis is

cholesterol

acyl

terol

of

cholesterol

HMG-CoA

reductase

diurnal

rhythm

Zucker

rats

lated

the

HMG-CoA

Our in to

was

HMG-CoA

reductase

In in

lack

most the

of coordinated

feeding

expressed

as percentages

feeding

the

pattern

Zucker

lean

might

ble

that

is

a characteristic

activity. reductase 7ct-hydroxylase

might

suppress

activity

could

be stimulated

phosphatase

of cholesterol

This

mechanism

activity

in

activity

7o-hydroxylase

hyperinsulinemia

suggested (21). was reported

note

obese

are

regu-

report,

however, (18).

Light

the

and

in

The

and dark

no significant out

that

rats

abolished

Zucker

differ-

possibility

rats.

variation

insulin

that

to explain

possi-

the

enzyme

cholesterol

7a-

kinase

and

might

promote

the

suppress the

The suppression recently

is

protein

level

and hence

It of

reconstituted

by CAMP-dependent High

regula-

activities.

obese

the

diurnal

to

(19).

ruled

diurnal

that

(20).

has been

by insulin

of

the

two activities

showed

CoAcholes-

activities

recently

of

low

Zucker

One recent

of enzyme

acyl

of diurnal

also

of these

This

removal

at

female

enzyme

(2,17).

groups.

reported

dephosphorylation

two

of 24 hr intake

recently

by alkaline

obese

examined

rhythmicity

the

interesting

was

these

We have

hydroxylase

in

activity

were

and obese

hyperinsulinemia

activity.

inhibited

rats

affect

Hyperinsulinemia

increased

is

increased

and

loss

and

degradation.

while

the

It

regulation

of

from

sacrificed

from

and

shown).

a consistent

rats

result

same direction

patterns

between

Zucker

instances,

not

cholesterol

revealed

lean

microsomes

result

reductase

activity.

activity

feeding

ences

be the

both

liver

could

vari-

Cholestyramine

7a-hydroxylase

results

obese

in

(data

decreased

cholesterol

7cy-hydroxylase

(16).

rats

by

this

coordinately

revealed

Zucker

regulated by

in

either

or

activity

of

altered

uptake

transferase.

We interpreted

content

dietary

regulated

7o-hydroxylase

high. tion

is

present

diurnal

cholestyramine.

P-450

obese

Thus,

cycle. was

with

was not

in

light

activity

treatment

content

synthesis,

cholesterol

of the

cytochrome

Hypercholesterolemia

De -- novo

middle

7cl-hydroxylase

rats

cholesterol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(22).

this

increased of

enzyme DMG-CoA

cholesterol

Vol. 150, No. 2, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgments This research was supported by a NIH grant, GM 31584, and Research and Academic Challenging Awards from the State of Ohio to J.Y.L.C. and a Biomedical Research Support RR 05806-07 to J.A.F. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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