Activities of some key enzymes of carbohydrate, ketone body, adenosine and glutamine metabolism in liver, and brown and white adipose tissues of the rat

Vol. 138, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 687-692

July 31, 1986

ACTIVITIES OF SOME KEY ENZYMES OF CARBOHYDRATE, KETONE BODY, ADENOSINE AND GLUTAMINE METABOLISM IN LIVER, AND BROWN AND WHITE ADIPOSE TISSUES OF THE RAT Greg Cooney, Rui Curl, Andrew Mitchelson, Philip Newsholme, Morag Simpson and Eric A. Newsholme Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXI 3QU, U.K. Received May 27, 1986 In general, the activities of enzymes in brown adipose tissue (BAT) are more similar to those in white adipose tissue than those in liver. Thus the activities of the glycolytic enzymes hexokinase and 6-phosphofructokinase are high but those of glucose 6-phosphatase and fructose bisphosphatase are non-detectable in the two adipose tissues. The activity of HMG-CoA synthase was non-detectable in BAT indicating that this tissue, unlike liver, cannot produce ketone bodies from fatty acid oxidation but, since the tissue possesses a high activity of HMG-CoA lyase, it might produce ketone bodies from leucine catabolism. The findings suggest that "metabolically" brown adipose tissue can be classified better as an adipose tissue than as a peripheral liver. A high activity of 3-oxoacid CoA transferase but a non-detectable activity of 3-hydroxybutyrate dehydrogenase suggests that BAT can utilise acetoacetate but not 3-hydroxybutyrate for heat generation during cold exposure plus starvation. © 1986AcademicPress, Inc.

INTRODUCTION: Brown adipose tissue (BAT) consists of small cells which contain

many mitoehondria and many lipid droplets. In contrast, white

adipose tissue contains large cells with few mitochondria and

usually

a single lipid droplet. There is no doubt that BAT is involved in heat production, particularly in hibernating animals during arousal and very for

in

young animals which do not shiver (I). The biochemical mechanism the

formation

mitochondria

of

from

heat

is

now

protein

established.

As

with

other tissues, the oxidation of fuels leads to the

generation of a proton-motive force but specific

well

in

the

BAT

mitochondria

possess

a

inner membrane that transfers protons from

outside to inside the inner mitochondrial membrane without concomitant ATP

formation

(2).

Of

particular

importance is the fact that this

proton-carrier activity of the protein can nucleotides

and

be

regulated

by

adenine

fatty acids of specific chain-length (3). This means

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Vol. 138, No. 2, 1986

that the tissue mitochondria

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

may

for

be

long

able

to

function

without

uncoupling

periods of time when, presumably,

its

it could be

considered to be a "normal ~ tissue. Indeed it has been suggested

that

the rate of uptake of glucose by this tissue may increase dramatically after a meal, in response to insulin, so that the tissue may

play

an

important role in the regulation of the blood glucose level (4). Thus, although BAT was presumably called adipose tissue because of its lipid

content,

it

could

high

be metabolically and functionally distinct

from white adipose tissue.

The role of white adipose tissue is considered to be the storage of

triacylglycerol which is mobilised as fatty acids under conditions

of starvation, stress, sustained exercise and injury (5). However, the

more

primitive

fish

(e.g.

elasmobranchs),

triacylglycerol is

stored almost exclusively in the liver and a discrete only

evolved

stored

triacylglycerol

as

bodies (7). For this reason it tissue

adipose

tissue

in more advanced fish (e.g. teleosts)(6). The available

evidence suggests that the liver of elasmobranchs the

in

could

be

considered

does

not

mobilise

long chain fatty acids but as ketone seemed

to

be

possible

that

brown

adipose

a primitive liver rather than a

different form of adipose tissue.

Mammalian particularly

liver

has

characteristic

some of

metabolic that

functions

tissue,

that

are

such as capacities for

both glycolysis and gluconeogenesis and for ketone body formation. For this

reason,

it was considered important to investigate that maximum

activities of key enzymes of these pathways in brown and white adipose tissue

and

liver.

In

addition,

for

the

purpose

of inter-tissue

comparison with a view to providing more information on the role

of

brown

metabolism,

adipose

tissue~

activities

of enzymes of adenosine

some key enzymes of the Krebs cycle and

also been measured.

688

metabolic

glutamlnase

have

Vol, 138, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHODS: Adult male rats were obtained from OLAC (76) Ltd., Bicester, Oxon, U.K. and were maintained at 23 + l°C under a 12h llght-12 h dark cycle. During this time they were fed on Oxold rat and mouse breeders diet (H.C. Styles, Bewdley, Worcs., U.K.). Rats were killed by cervical dislocation and the liver, interscapular brown adipose tissue and epidldymal white adipose tissue were removed. Tissues were immediately extracted in extraction medium and activities measured as described previously for enzymes of glycolysis and gluconeogenesis (8,9), enzymes of ketone body metabolism (I0,Ii) enzymes of adenosine metabolism, the Krebs cycle and glutamlnase (12,13,14). Activities are expressed as nmol/min per mg protein. RESULTS AND DISCUSSION:

The

activities

of

gluconeogenlc enzymes are similar in white and and

quite

distinct

the

glycolytic

brown

adipose

and

tissue

from those in the liver: thus, the actlvltles of

hexokinase and especially 6-phosphofructokinase are very high

in

the

adipose tissues - considerably higher than those of the liver, whereas those of glucose 6-phosphatase and fructose 1,6-blsphosphatase are not detectable in both adipose tissues but are present in the liver (Table i). Similarly, the activity of glucokinase is high in liver but is not detectable

in

either

adipose tissue (Table I). It is of interest to

note that pyruvate carboxylase and activities

are

present

in

all

probably present in the adipose process

(5)

rather

than

a

phosphoenolpyruvate three

tissues part

carboxyklnase

tissues;

these

as

of

part

enzymes are

the

llpogenic

of gluconeogenesls. There is more

similarity in the pattern of activities of the adenosine

metabolising

enzyme between white and brown adipose tissue than either of these two tissues and liver (Table i). The high activity of in

the

5"nucleotidase

comparison to those of the kinase and deamlnase in the liver is in

contrast to the activities of these enzymes suggesting

the

the

adipose

tissues

role

of

the nucleotldase and indeed adenosine

might be different in liver

to

that

possible

that

in

in

the

adipose

tissues:

one

role in the adipose tissues is modulation of the sensitivity

of glucose utillsation and llpolysls to insulin (15, 16).

Of the enzymes necessary for production of acetyl-CoA,

the

activity

of

ketone

bodies

from

HMG-CoA synthase was not detectable in

689

Vol. 138, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I - Maximum activities of some key enzymes of carbohydrate, ketone body adenosine and glutamine utilisation in liver, white adipose tissue and brown adipose tissue Enzyme activities (nmol/min per mg protein at 25°C)

Enzyme

Liver

White adipose tissue

Glueokinase

21.5 ± 2.77

Hexokinase

4.4 ± 0.23

Glucose 6-phosphatase

<0.1

49.7 ~ 2.01

6-Phosphofructokinase

9.7 ± 0o54

Fructose bisphosphatase

26.8 ± 1.39

l>gruvate kinase

481"

Fyruvate ca~bo~lase Phosphoenolpyruvate

61.6 ± 5.5

<0.1

<0.1

41.8 ~ 5.80

67.0 ! 3.0

<0.1

<0.1

90.8 ± 8.87

403 ± 4.51

19.6 ± 0.49

36.8 ± 0.62

4.4"

4.6 ~ o.~h

13.1 ± 0.35

6.5 ± 1.2

2.2 ± 0.31

31.9 ! 1.10

2.1 ! 0.25

<0. I

carbo~ykinase dehydrogenase

~1.4 ! 7.9

3-0xoacid CoA dehydrogenase

3.0 ~ 1.52

Aoetoaoetyl-CoA thiolase

81.3 ± 8.7

HMG-CoA synthase

< 0.1

31.9 ± 2.61

74.6*

Glucose 6-phosphate dehydrogenase 3-Hydroxufbut~ate

Brown adipose tissue

21.1 ! 0.85

204 ! 42

18.1 ! 0.32

182 ~ 38

2.7 ± 0.57

-

< 0.1

HMG~CoA lyase

36.9 ± h.3

5'-Nuoleotidase

82.3 +

11.4 ~ 1.31

38.2 ± 9.3 3.2 ± 0.35

Adenosine deaminase

10.9 +

18.1 ± 1.81

14.2 ~ 0.57

Adenosine kinase

28.1 +

0.27 ± 0.0h

0.09 ± 0.0I

Citrate synthase

33.8 ± 2.12

38.2 ± 3.06

380 ~ 13.7

0xoglutarate dehydrogenase

3~.0 ~ 1.8

Glutaminase

13.2 ± 1.9

150 ~ 10.5 24.9 ± 0.8

39.0 ~ 3.2

The methods for measuming enzyme activities are given in the Methods section. presented as means ~ S.E.M. for at least six separate animals.

Results are

Data taken from previous

work *(8) +(12).

brown adipose

tissue but that of HMG-CoA

the liver. This demonstrates the liver, However,

is unable

could

possesses activity

in the metabolism produce

ketone

a high capacity to utilise

cold

exposure;

rat

to that

in

in contrast

from

fatty

acids.

of lyase is unclear: in

which

to

case

it

this

from leucine.

In addition,

BAT

CoA transferase

but a v e r y

low

dehydrogenase acetoacetate

brain

tissue,

bodies

leuclne;

hodles

he an important since

of

of 3-oxoacid

of 3-hydroxybutyrate

may

ketone

of the high activity

a high activity

Acetoacetate

that brown adipose

produce

the significance

may be involved tissue

to

lyase was similar

which suggests but

not

3-hydroxybutyrate.

fuel for BAT during can

690

use

either

that it has

starvation acetoacetate

or

Vol. 138, No. 2, 1986

3-hydroxybutyrate

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as

a

fuel

during

starvation

(17),

lack

hydroxyhutyrate dehydrogenase in brown adipose tissue may be

a

of

means

of conserving 3-hydroxyhutyrate particularly for the brain during this condition.

There is a high activity of the enzymes of the brown

adipose

tissue

(e.g.

citrate

Krebs

synthase

and

cycle

oxoglutarate

dehydrogenase, Table I). These activities are at least lO-fold than

those

in

higher

in liver or white adipose tissue: this is consistent with

the high oxidative capacity of this generation.

tissue

which

is

used

in

heat

It has been shown previously that the maximum activity of

oxoglutarate

dehydrogenase

provides

a

quantitative

index

of

the

maximum capacity of thermogenesis in this tissue (4).

The

activities

of

glutamlnase in both white and brown adipose

tissue are higher than that in liver which suggests that these tissues have a high capacity to utillse glutamine; the significance of this is unclear.

ACKNOWLEDGEMENTS: (Fundac~o

R.C. was a recipient of a

Fellowship

from

FAPESP

de Amparo a Pesquisa do Estado de S~o Paulo) and P.N. and

M.S. were recipients of SERC Training Awards.

REFERENCES : I. Smith,R.E. & Horwitz, B.A. (1969). p hZ§iol.Rev. 49, 330-425 2. Nicholls, D.G. (1979). Biochim.Biophys.Acta 549, 1-29 3. Rial, E., Poustle, A. & Nicholls, D.G. (1983).Eur.J.Biochem. 137, 197-203 4. Cooney, G.T. & Newsholme, E.A. (1984). Trends Biochem.Sci. 9, 303-305. 5. Newsholme, E.A. & Leech, A.R. (1983). Biochemistry for the Medical Sciences, John Wiley & Sons Ltd., Chichester, England. 6. Love, R.M. (1970). The Chemical Biology of Fishes, Academic Press, London and New York. 7. Zammlt,V.A. & Newsholme, E.A.(1979). Biochem.J. 184, 313-322. 8. Dohm, G.L. & Newsholme, E.A. (1983). Biochem.J. 212, 633-639.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

9.Stanley, S.C.~ Dohm, L., McManus, B. & Newsholme, E.A. (1984). Biochem.J. 224, 667-671. I0. Zammit, V.A., Beis, A. & Newsholme, E.A. (1979). FEBS Lettr. 103, 212-215 Ii. Beis, A., Zammit, V.A. & Newsholme, E.A. (1980). Eur.J.Biochem. 104, 209-215. 12. Arch, J.R.S. & Newsholme, E.A. (1978). Biochem.J. 174, 965-977 13. Cooney, G.T., Taegtmeyer, H. & Newsholme, E.A.(1981).Bioehem.J., 200,701-703. 14. Curthoys, N.P. & Lowry, O.H. (1973). J.Biol.Chem. 248, 162-168. 15. Schwabe, U., Sch6nh~fer, P.S. & Ebert, R. (1974). Eur.J.Biochem., 46, 537-545. 16. Green, A. & Newsholme, E.A. (1979)o Biochem.J. 180, 365-370. 17. Hawkins, R.A., Williamson, D.H. & Krebs, H.A. (1971).Biochem.J. 122, 13-18.

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