Regulation of the multiple molecular forms of rat liver glucose 6-phospnate dehydrogenase by insulin and dietary restriction

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 28, 1985

Pages 136-142

Regulation of the Multiple Molecular Forms of Rat Liver Glucose 6-Phospnate Dehydrogenase by Insulin and Dietary Restriction 1 Ralph N. Martins*,

Gilbert B. Stokes§

and Colin L. Masters*

*Laboratory of Molecular and Applied Neuropathology Neuromuscular Research Institute, D e p a r t m e n t of Pathology, and §Department of Biochemistry, University of Western Australia, Nedlands, Western Australia 6009

Received January 4, 1985

Insulin treatment of v i r g i n female rats increased the hepatic a c t i v i t y of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase to levels 3.4 and 1.5 fold higher than controls. The increase in glucose 6-phosphate dehydrogenase a c t i v i t y was a t t r i b u t e d to increased a c t i v i t y of a l l three dimer species. Thus dimer bands, 1, 2 and 3 of i n s u l i n - t r e a t e d animals were 5, 3 and 2-fold higher respectively than controls. The a c t i v i t y of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase decreased with fasting to 55% and 72% respectively of controls. The decrease in glucose 6-phosphate dehydrogenase a c t i v i t y r e f l e c t e d a lower a c t i v i t y of dimer bands 2 and 3 only, which were 62% and 39% of control a c t i v i t y respectively a f t e r three days fasting. A s h i f t towards band 1 was observed under both conditions of starvation as well as under conditions of insulin treatment. ®198SAo~d~ioPres~,~nc.

The rat liver hexose monophosphate dehydrogenases, glucose 6-phosphate dehydrogenase

(D-glucose

6-phosphogluconate

6Zphosphate-NADP + dehydrogenase

oxidoreductase,

EC

(6-phospho-D-gluconate:NADP+

1.1.1.49)

and

oxidoreductase

(decarboxylating), EC 1.1.1.4tt) have been demonstrated to change in activity under various metabolic and hormonal conditions (1,2,3,5,6).

The male rat model has been

used in most of the experimental studies on the nutritional and hormonal regulation of glucose 6-phosphate dehydrogenase activity and little information is available on the responses in the female rat where basal activity is about double that observed in male rat liver. Rat liver glucose 6-phosphate dehydrogenase has been shown to occur as at least 6 different activity forms (2).

It has not been established whether these molecular

/Supported in part by Grants from the National Health and Medical Research Council of Australia, and the Telethon Research Foundation. *To whom c o r r e s p o n d e n c e should be a d d r e s s e d . 0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

136

Vol, 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

forms are d i f f ere n t gene products, though t h e re is some evidence to suggest that they represent

different

molecular

forms

of

the

same

enzyme

protein (2, 3 and 5).

However, Hori and Matsui (8) have shown under in vitro conditions that one particular form of glucose 6-phosphate dehydrogenase is s e l e c t i v e l y inhibited by the sex steroid dehydroepiandosterone.

In this paper we describe the response of specific molecular forms of hepatic glucose 6-phosphate dehydrogenase to insulin levels in the f e m a l e rat

that were raised by

insulin administration or lowered by fasting.

MATERIALS AND METHODS Materials.

D-Glucose

6-phosphat%

6-phosphogluconate,

NADP +,

Trizma

base,

nitroblue t e t r a z o l i u m and phenazine methosulphate were purchased from Sigma (St. Louis, Mo).

All other materials were of r e a g e n t grade and obtained locally.

Rats of the V¢istar strain were obtained from the Animal Resources C e n t r e of Western Australia.

Fourteen week old virgin rats were used in all experiments.

They were

housed in an airconditioned room under a controlled 12h light (7.15 am - 7.15 pro) 12 h dark

(7.15 pm

pelleted

diet~

- 7.15

am) cycle.

All animals were fed ad libitum a c o m m e r c i a l

(Milne

Feeds

Ltd.~

Pty.

Perth,

W.A.)

containing

3-6%

fat

(60%

polyunsaturated) and about 68% carbohydrat% for seven days prior to c o m m e n c e m e n t of the e x p e r i m e n t a l period.

Animal t r e a t m e n t and tissue preparation. c o m m e n c e m e n t of experimentation. was starved for 72h. saline

every

12h.

Units/lOOg/12h). rapidly

excised,

Rats were divided into t h r ee groups at the

Groups A and B were fed ad libitum and group C

Group A and C were injected subcutaneously with physiological Group

B

was

injected

with

protamine

zinc

insulin

(3

Animals were sacrificed between 10am and 12 noon. The liver was weighed and

homogenized in a cold Perspex

homogenizer of the

P o t t e r - E l v e h j e m type with t h r e e volumes of 10mM T r i s - H C l , bufler pH 7.# containing 0.25M sucrose and lmM EDTA.

The homogenates were i m m ed i at el y centrifuged at

130000 x g for 60 rain, and the high speed supernatant fraction was collected. 137

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Assays of enzyme activities.

Glucose 6-phosphate dehydrogenase and 6-phosphogluco-

hate dehydrogenase a c t i v i t i e s were measured s p e c t r o p h o t o m e t r i c a l l y at 25°C according to the procedure of Rudack et al. (1).

6-phosphogluconate dehydrogenase was assayed

in the presence of 0.6ram 6-phosphogluconate and 0.9ram

NADP +, pH 7.6.

The

combined a c t i v i t y of both dehydrogenases was measured in the presence of 0.6ram 6-phosphogluconate, 0.9mM NADP + and 2ram glucose 6-phosphate at pH 7.6. 6-phosphate

Glucose

dehydrogenase a c t i v i t y was e s t i m a t e d as the d i f f e r e n c e between

the

combined a c t i v i t y and the 6-phosphogluconate dehydrogenase activity. The amount of the high speed supernatant f r a c t i o n assayed for enzyme a c t i v i t y corresponded to 5rag of liver.

One unit of enzyme a c t i v i t y is that amount of enzyme which c a t a l y s e s the

reduction

of

1 [1mole of NADP + per minute under the conditions of the assay.

Enzyme a c t i v i t y is expressed as U/g tissue wet wt. Electrophoresis.

Electrophoresis of the high-speed liver supernatant fractions was

performed within

3h of preparation using a 1.5mm thick slab gel containing 10%

(w/v) a c r y l a m i d e and 0.26% (w]v) b i s a c r y l a m i d e polymerized in the presence of 0.017% (w/v)

ammonium

acrylamide

persulphate

stacking

gel was

and used

0.033%

(v]v)

tetramethylene

diamine.

A 75%

and samples were applied d i r e c t l y in a 5mM

T r i s - H C l , pH 7.# loading buffer containing 20% (w/v) sucrose and bromophenol blue as the tracking dye.

The elctrophoresis buffer was 25 mM Tris, and 200 mM glycine, pH

8.3. NADP + (10 [JM) was included in the cathode buffer. Samples were e l e c t r o p h o r e s e d at a constant current of 15 mA/gel and then stained for a c t i v i t y in pH 8.0.

50 mM

Tris-HCl, containing 0.58 mM glucose 6-phosphate, 0.13 mM NADP +, 10 mM MgCI2, 33 [~M phenazine

methosulphate

and 0.1 mM nitroblue t e t r a z o l i u m .

incubated in this staining solution at #°C for 16h. immersing the gel in 7% a c e t i c

The gel was

The reaction was then stopped by

acid, a f t e r which it was photographed and scanned

using a GS 300 gel scanner (Hoeffer Scientific Instruments, San Francisco) ( F i g u r e 1 ) .

RESULTS In this study the a c t i v i t y of both hexose monophosphate dehydrogenases d e c r e a s e d

in

the liver to 55% and 72% of controls respectively when rats were starved for 72h (P<.01).

The a c t i v i t y of glucose 6-phosphate dehydrogenase and 6-phosphogluconate 138

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Non-denaturing gel electrophoresis Activity staining of rat liver glucose 6-phosphate dehydrogenase on a 7.5% polyacrylamide slab gel. Lanes) 1, 2, 3 contain samples £rom control animals. Lanes 4,5,6 contain samples from animals treated with insulin for 72h. Lanes 7)g,9 represent samples from rats fasted for 72h. An average of 750+_.50 pg total protein was loaded in each well. Electrophoretic conditions and enzyme activity staining are described in methods.

dehydrogenase

increased

values respectively supernatants

(P ~

0.01) (Table 1).

demonstrated

(as d e t e r m i n e d

in the i n s u l i n - t r e a t e d

group to 2##% and

150% of c o n t r o l

Non-denaturing gel electrophoresis of high speed

that total liver glucose 6-phosphate dehydrogenase activity

densitometrically)

increased

to

258% of c o n t r o l s (P<.01). H o w e v e r ,

though s t a r v a t i o n caused a d e c r e a s e in ~otal a c t i v i t y , this d e c r e a s e was not s i g n i f i c a n t (P > 0.05) (Table 2A). The insulin-promoted increase in liver glucose 6-phosphate dehydrogenase a c t i v i t y was due to i n c r e a s e s

in a c t i v i t y of all t h r e e d i m e r a c t i v i t y bands.

However,

d e n s i t o m e t r i c scanning of t h e e n z y m e bands f r o m s t a r v e d animals r e v e a l e d t h a t none of t h e bands,

1, 2 or 3 was s i g n i f i c a n t l y a l t e r e d when c o m p a r e d with c o n t r o l values.

To d e t e r m i n e

whether

t h e r e l a t i v e ratios of t h e bands 139

1, 2 and 3 w e r e a l t e r e d by

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1. Changes in hepatic G6PD a c t i v i t y under d i f f e r e n t e x p e r i m e n t a l conditions

E n z y m e activity (Units/g tissue wet wt.)

Treatment

6PGD Control

G6PD

6.60+.35 a

3.g0+.39 d

Fasted

$.76+0.3 b

1.87+0.17 e

Insulin

9.98+ 1.07 c

8.3+ 1.#5 f

Each value r e p r e s e n t e d the mean + SE of 8 animals. The d i f f e r e n c e s b e t w e e n (a) and (b), (a! and (c), (d) and (e), and (d) a n d (f) are all statistically significant (P<0.01).

either

insulin treatment

ages of total activity. decrease

or starvation,

we have expressed

Only band 3 from insulin-treated

band activities animals exhibited

as percenta significant

(Table 2B).

DISCUSSION In t h i s p a p e r , w e c h o s e to r e s t r i c t the

dimer

molecular

forms.

TABLE 2. A.

Quantitation

Band activity in mg.

Control

our s t u d y o f g l u c o s e 6 - p h o s p h a t e

Higher aggregates

were observed

of t h e molecular forms of measured d e n s i t o m e t r i c a l l y Insulin T r e a t e d

liver

dehydrogenase

on t h e gels~ b u t t h e i r

G6PD

activity

Band 1

i#+#.6 a

68+24 b

13.7+1# c

#7+15.7 d

130+22 e

29+12.7 f

Band 3

56+20 g

105+12.7 h

22+7 i

Comparison of

liver

G6PD molecular

as

Fasted

Band 2

B.

forms

as percentage of t o t a l

activity Insulin T r e a t e d

Band a c t i v i t y % of total

Control

Band 1

12.2+2.1 j

22+7.3 k

16.7+14.71

Band 2

40+1 m

42.6+4 n

~6.5+4 °

Band 3

~7.6+1.5 p

35+7 q

36.8+10 r

Fasted

Each value r e p r e s e n t s the mean + S.D. of 3 rats, (a) is significantly d i f f e r e n t from (b) (P<.05) but not from (c); (d) is signficantly d i f f e r e n t from (e) (P<.01) but not from (f) (P>.05); (g) is significantly d i f f e r e n t from (h) (P<.05) but not from (i) (P>.05); (j) is not significantly d i f f e r e n t from (k) or (l) (P>.05). (m) is not signficantly d i f f e r e n t from (n) or (o) (P>.05). (p) is signficantly d i f f e r e n t from (q) (P<.05) but not from (r) (P>.05). 140

to

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity never exceeded 5% of the total activity~ in accordance with earlier reports

(2,3). Our

findings of

decreased

activity

of

the

hexose monophosphate dehydrogenases

following fasting and the increased activity following insulin t r e a t m e n t of the female rat

is

in

agreement

with

previous

reports

for

the

male

rat

(1,4).

In our

electrophoresis experiments we always used similar amounts of sample protein to prevent

any

protein

concentration-dependent

shift

dehydrogenase molecular forms that might occur.

in

the

glucose

6-phosphate

The results demonstrate that the

insulin-promoted increase in glucose 6-phosphate dehydrogenase activity is due to an increase in all three 6-phosphate

dimer bands.

The changes in the

intensity of the glucose

dehydrogenase activity bands both in response to fasting and insulin

t r e a t m e n t , are similar for all the three dimer bands.

However, there does appear to

be a decrease in band 3 and an increase in band 1 for both the insulin treated and the fasted animals~ though only the change associated with band 3 of the insulin treated group was significant (Table 2B).

Taketa and Watanabe (1971) demonstrated that

sulfhydryl reagents can alter

the

distribution of the dimer species (5).

They have also shown that band 1 activity

increased in aged preparations of rat

liver glucose 6-phosphate dehydrogenase as

compared to freshly prepared rat liver glucose 6-phosphate dehydrogenase. Recently, Grigor

(3) confirmed

6-phosphate

these

earlier

observations for

m a m m a r y gland glucose

dehydrogenase and proposed that band 1 and band 3 represent fully

oxidized and

fully reduced

forms

of

represents a partially oxidized form (3). susceptible

rat

to

proteolytic

the enzyme respectively, and that

band 2

Grigor (3) also showed that band 1 was most

inactivation and

6-phosphate dehydrogenase activity decreases

that

when

mammary

gland

glucose

on weaning, a c o n c o m i t a n t selective

shift in the dimer banding p a t t e r n towards band 1 occurred.

Chang et al. (6) observed

a slight shift towards band 1 in the fasted refed induced male rat.

Their finding and

that of ours with the insulin induced female rat may be explained by earlier reports that the induction of glucose 6-phosphate dehydrogenase results in an increase in both the rate of synthesis as well as the rate of degradation of the enzyme (1,7). 141

Taken

Vol. 127, No. 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

together, our results lend support to Grigor's proposal (3) that a shi:[t in the dimer banding pattern towards band 1 may be an early event in the degradation of this enzyme.

REFERENCES 1.

Rudack, D., Chisolm, E.M., and Holten, D. (1971) 3. Biol. Chem. 246, 12#9-125#.

2.

Holten, D. (1972) Biochem. Biophys. Acta 26g, #-12.

3.

Grigor, M. (198#) Arch. Biochem. Biophys.

#.

Weber, G. (1963) in Advances in Enzyme Regulation (weber, G.,

229, 612-622.

ed) Vol. 1, pp 1-20, Permagon Press, London. 5.

Taketa, K., and Watanabe, A. (1971) Biochim. Biophys. Acta 235, 19-26.

6.

Chang, H., Holten, D. and Karin, R. (1979) Can. 3. Biochem. 57, 396-#01.

7.

Morikawa, N., Nakayama, R. and Holten, D. (198#) Biochem. Biophys. Res. Commun. 120, 1022-1029.

8.

Hori, S.H., and Matsui, 3. (1968) 3. Histochem. Cytochem. 16, 62-63.

142