Effect of streptozotocin diabetes on some urea cycle enzymes

Vol. 106, No. 1, 1982 May 14, 1982

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 37-43

EFFECT OF STREPTOZOTOCIN DIABETES ON SOME UREA CYCLE ENZYMES A. Jordd,

M. Gomez*,

J.

Cabo* and S. Grisolfa

Instituto de Investigaciones CitolBgicas de la Caja de Ahorros de Valencia; and *Departamento de Bioquimica de la Facultad de Farmacia de Valencia. Valencia, Spain. Received February 25, 1982 Streptozotocin induced diabetes in rats increased the activities of the three mitochondrial enzymes, carbamylphosphate synthetase, ornithine transcarbamylase and N-acetylglutamate synthetase, but not of the cytosolic N-acetylglutamate deacylase. Levels of both N-acetylglutamate and arginine, which are activators of carbamylphosphate synthetase and N-acetylglutamate synthetase respectively, increased in diabetes. These results serve to explain the increase both of mitochondrial citrulline and urea formation in hepatocytes and the increased urea excretion in diabetes. The increase

in urea

ance in diabetes amino acids

for

excretion

are attributed

and the

mainly

gluconeogenesis

negative

to increased

and to decreased

nitrogen

bal-

utilization

protein

of

synthesis

(1,2). As a catabolic being

regulated

cycle,

only

urea

by the

availability

ses in the ammonia load would synthesis

(4).

(AG) , which synthetase varies for

(CPS),

AG synthesis

subject

is

is

to explain

in all

state

strongly

regarded

substrates

result

activator

present

is generally of

therefore

fails

the metabolic

(3).

in changes

in urea

why N-acetylglutamate (5) of carbamylphosphate

ureotelic

(6,7,8).

dependent

vertebrates,

and

The enzyme responsible on the

that

to close

emerges from these

concentration

regulation,

although

observations the metabolic

as

Increa-

of

(9).

The picture is

view

is an obligatory

with

arginine

This

synthesis

is thatCPS advantages

Abbreviations: CPS: carbamylphosphate synthetase, OTC: ornithine transcarbamylase, AG: N-acetylglutamate, AG-synthetase: N-acetylglutamate synthetase, AG-deacylase: N-acetylglutamate deacylase. 0006-291X/82/090037-07$01.00/0

Vol. 106, No. 1, 1982

of

such fine

further

regulation

evidence

the level

BIOCHEMICAL

for

AND BIOPHYSICAL

are not well the

perimental

understood

regulation

of the

of CPS, by demonstrating

RESEARCH COMMUNICATIONS

increased

as yet.

synthesis

We present

of urea

levels

at

of AG in ex-

diabetes.

MATERIALS AND METHODS Male Wistar rats (200-250 g) were fed "ad libitum" a standard diet containing 18% protein. Diabetes was induced by a single intraperitoneal injection of 65 mg/Kg of Streptozotocin (10). The rats were sacrificed at the same time of day by decapitation. The tissues were homogenized with an Ultra-turrax under established conditions (11) and diluted appropriately with bi-distilled water. The mitochondria were prepared as described by Hogeboom et except that mannitol was used instead of sucrose to avoid al (121, zterference in the determinations of citrulline and urea (13). Hepatocytes were isolated according to Berry and Friend (14). Mitochondria were incubated as described by McGivan et -al (15) in 25 ml closed vials. Hepatocytes were incubated according to Briggs and Freedland (16). Citrulline and urea were assayed according to Hunninghake and Grisolfa (17). CPS and ornithine transcarbamylase (OTC) activities were determined according to Schimke (18) in whole liver homogenate. Nacetylglutamate deacylase (AG-deacylase) was assayed according to Grisolca et al (19). N-acetylglutamate synthetase (AG-synthetase) was deterzn??? according to Shigesada and Tatibana (20) in the (NH ) SO precipitate of supernatant from sonicated mitochondria whi$h2 had 4 been centrifuged at 105,000 x g. AG was determined as described by Shigesada and Tatibana (9), and arginine was determined according to Saheki -et al (21). Blood glucose assay was according to Werner et by a -- al (22) and protein biuret-deoxycholate method (23). All results have been submitted the degree of significance calculated an Olivetti-101 programmer.

to statistical by Student's

analysis and t test, using

RESULTS As anticipated, liver urea

diabetic

and body weight excretion

(Table

cycle

hepatocytes

isolated

hepatocytes

from control

capacity

enzymes.

of

excreted We expected increased

However,

from diabetic

of ammonia (Table of

I).

was the result

to the urea

tions

rats

rats I).

the urea cycle

rats

the

in diabetic

rats.

increased

ammonia availability

produced

indicated

38

that

we were surprised

when exposed This

more urea and hadlower

to

urea

to identical a higher Moreover,

find that

faster

than

concentra -

functional intact

mi-

Vol. 106, No. 1, 1982 Table

I.

BIOCHEMICAL

AND BIOPHYSICAL

Effect of diabetes on blood glucose, citrulline synthesized urea excretion, dria and urea synthesized in isolated

Body

weight

Liver

Citrulline isolated

632

It 33

p (

210

+ 20

175

+ 31

p < 0.001

A

P < 0.01

tochondria more

freshly

rapidly

process the

rate

the

result

drial

927

k 32

1.3

It 49

p 6 0.001

4.8

+

0.4

7.7

k

0.6

p (

3.5

It

0.7

7.4

k

0.8

p < 0.001

is

of

operation of

essential

and in

the the

by

intact

which AG.

As

in shown

in

Of

saturating

activity

(5).

part was

of

the

of

depends Table

on II,

the

and

(Table

only

partly of

two the

latent these

rate by

the

amounts

of

CPS and

AG were

as

the

II,

intact

miand

AG.

citrulline levels

enzymatic elevated

a

which

organelles

of

determined

39

is

Table

of

is

both

mitochon-

in

concentration the

is

first

in

of

assay,

control

viva",

illustrated

disruption

a

operative

AG "in

is

I), to

OTC,

enzymes,

is

a saturating of

citrulline

synthesis

two

levels

by

mitochondria

in

is

CPS

CPS activity

revealed

conditions

turn

these

This

rats

Citrulline

of

addition

non

presence

Under thesis

the

action

produced

circumstances,

cycle.

0.001

and liver in nmol/ are ex-

rats

control

most

urea

cycle.

and

that

tochondria, assay

the

for

shows

the

sequential

of

of

under

of

the

diabetic from

believed,

active,

consequence

from

mitochondria

which

less

which

isolated

than

enzymes

much

of

6.9

Glucose is expressed in mg/lOO ml of blood; body in g; urea excretion in mg/24 hours: citrulline protein; urea in pmol/min/g wet weight. Results as mean f SD of 8 rats.

weight min/mg pressed

CPS,

1.2

0.001

in

Urea synthesized in isolated hepatocytes

is

k

575

synthesized mitochondria

test

* 10

7.8

urea

Student's

157

weight

Urinary

body and liver weight, urinary in isolated liver mitochonhepatocytes of rat.

DIABETIC

NORMAL Glucose

RESEARCH COMMUNICATIONS

synof

protein

active and

in

the

Vol. 106, No. 1, 1982 Table

II.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Effect of diabetes on carbamylphosphate synthetase(CP.51, ornithine transcarbamylase(OTC), N-acetylglutamate synthetase(AGsynthetase) and N-acetylglutamate deacylase(AG-deacylase) activities and on the N-acetyl-glutamate(AG) and arginine levels in rat liver. NORMAL

CPS

303

OTC

10,550

DIABETIC

f

77

764

f

850

13,100

Student's

f

test

p < 0.001

58

f 1100

p<

0.05

p < 0.001

AG-synthetase

56.7

+

10.4

90

k

AG-deacylase

83.3

f

8.8

80

f

7.7

AG

29.5

Tt

7.3

68.1

f

12.7

p < 0.001

Arginine

69

t

3.5

f

83

p < 0.001

306

CPS, OTC, and AG-deacylase activities h/g of liver: AG-synthetase in nmol/h/g of in nmol/g of liver. Results are expressed rats.

of diabetic

liver

cle

enzyme,

rats;

increased

AG is synthesized ted

in mitochondria;

lase

located

(19).

changes

found

that

AG-synthetase

while

that

of AG-deacylase

of

in activity

may

AG-synthetase

second factor although

ent,

its

i.e.

ginine

levels

rats

(Table

of

postulated

can

account

may also

these

did

be

by the very

abundant

of AG might

two enzymes.

not

change

that

an

the

for

for

importance

AG-synthetase

is

stimulated

were

to

be

found

an enzyme also

higher

II). 40

cy-

re-

rats

was

II). in

the

levels

the

increased

cannot

be

by

arginine, the

therefore

in diabetic

increase

in

AG-deacy-

it

(Table

increased

loca-

When tested,

was increased

be invoked

quantitative

(20),

The level

activity

urea

fo Id).

AG may be split

in cytosol

it

intramitochondrial

by AG-synthetase

flect

Thus

( 1.2

N.S.

are expressed in umol/ liver: AG and arginine as mean f SD of 12.

OTC, the other s lightly

18.1

livers

activity of

AG. A

AG levels,

evaluated and of

at the

diabetic

presar-

Vol. 106, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION The importance

of AG in the

has been demonstrated in this

paper

diabetes

stress

this

directly

synthesize

point

affect

citrulline

explaining

remains rise

acid

teolysis chain

of this

AG is

of

metabolite. and was found

increased

by diabetes

causes include:

1) a

and subsequent

due to

stimulated

by accumulating

and 3) inhibition

to

to synthesize

lipolysis

in arginine,

by

and CPS

homogenate

the possible

of arginase

(25,26);

changes produced

of hepatocytes

liver

Some of

inhibition

described

of the mitochondria

excretion

2) a rise

amino acids

pro-

branched-

of mitochondrial

AG

(8). Liver

acetyl-CoA

and hepatic teolysis

without

(9),

(29),

of AG in the

ysis

and ureogenesis The increased synthesis

citrulline responsible is modulated

that urea

activity

(28).

according

appear excretion

by isolated changes

by changes

coordination

regulated

in part

can be correlated

hepatocytes

and also

mitochondria. is the

greater

in the AG levels, 41

and libera-

et -- al

of diabetes the

stimulating

synthesis

to Meijer

(8),

glucagon

on the concentrabetween

proteol

by glucagon withthe with

(8).

increase

the increased

One of the CPS activity,

and which

(27),

increasedpro-

of AG by either

or by increasing

by isolated

these

due to

level

may affect

in diabetes

perhaps

AG. The effect

liver,

synthesis for

the

since

mitochondrial

tion

in arginase

increase

activity

increase

increases,

increase

of glucagon

in urea

also

could

AG-synthetase

increases

concentrations

arginine

Arginine

tion

capacity

due to activation

oxidation;

or/and

efflux

the

in total

to be elucidated.

activity

and on AG-synthetase

The mechanism by which

in acetyl-CoA,

fatty

and show that

increased

AG has been measured

cycle

The experiments

and the ability

the

to be increased.

of urea

(4,8,9,13,20,24).

on the AG concentration,

activities,

urea,

control

mightbe

factors which related

Vol. 106, No. 1, 1982

to the

BIOCHEMICAL

increases

AND BIOPHYSICAL

in acetyl-CoA,

RESEARCH COMMUNICATIONS

AG-synthetase

and its

activator

arginine. All linemia occur

of the changes and/or

observed

hyperglucagonemia,

could

result

absolute

from either

or relative

hypoinsu(30),

which

in diabetes.

ACKNOWLEDGEMENTS

Dr.

We wish to thank Dr. V. Rubio for F. Thompson for critically reading

his helpful suggestions the manuscript.

and

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