The effect of hypophysectomy on rat liver gluocokinase activity and on plasma glucose, insulin and nonesterified fatty acid concentrations

287

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

26171

T H E E F F E C T OF HYPOPHYSECTOMY ON RAT L I V E R GLUCOKINASE ACTIVITY AND ON PLASMA GLUCOSE, INSULIN AND N O N E S T E R I F I E D FATTY ACID CONCENTRATIONS C. M. DAWSON* AND C. N. HALES

Department of Biochemistry, University of Cambridge, Cambridge (Great Britain) (Received February 26th, 1969)

SUMMARY

I. The relationship of rat liver glucokinase (ATP-D-glucose 6-phosphotransferase, EC 2.7.1.2 ) activity to plasma insulin level has been examined in hypophysectomized rats. 2. Glucokinase activity was found to be depressed by at least 5o % after hypophysectomy when compared with age, weight, sham-operated and pair-fed controls. 3. The plasma insulin level in hypophysectomized rats was found to be lower than in all control animals. 4. Attempts have been made to restore to normal the low glucokinase activity by hormone injections. 5. Thyroxine and growth hormone had no effect on either the glucokinase activity or the plasma insulin. 6. Hydrocortisone raised the plasma insulin in 12 h and the enzyme activity in 24 h. Administration of growth hormone with the hydrocortisone produced a more rapid response in the rise in plasma insulin, a change being detected 6 h after the injection. Glucokinase activity was raised by injection with both hormones, a slight rise was seen after 12 h. 7. Phosphofructokinase (EC 2.7.I.II ) and pyruvate kinase (EC 2.7.1.4o ) were also assayed in livers from hypophysectomized rats; pyruvate kinase activity was approx. 5o% of the control value whereas phosphofructokinase activity was unchanged by the operation.

INTRODUCTION

Rat liver glucokinase (ATP-D-glucose 6-phosphotransferase, EC 2. 7. I. 2) activity has been shown by several workers to be lower than normal in starved, alloxan diabetic 1-6, anti-insulin serum diabetic 7 and rats fed a low carbohydrate diet s-l°, all conditions in which the plasma insulin is below normal. Maintenance of normal glucokinase activity has been assumed to be dependent upon normal plasma concentrations of glucose and insulin n-13. * N6e C. M. Firth, present address: School of Biological Sciences, University of East Anglia, Norwich, England.

Biochim. Biophys. Acta, 184 (I969) 287-298

GLUCOKINASE ACTIVITY AFTER HYPOPHYSECTOMY

289

of supernatant (approx. I0/*1) had reached a minimum. No correction was made for any contribution of the hexokinases (EC 2.7.1.1 ) in the assay, since at least one of the isoenzymes is inhibited b y high glucose concentrations 19 and since on several occasions a higher rate at 0.5 mM than at I00 mM glucose has been observed on supernatant from hypophysectomized rats. The rate achieved in this assay after the initial lag period was identical to that obtained on dialysed supernatant after correction for a no-substrate blank rate. In all cases, 2/,moles of NADP + were reduced per/*mole of glucose phosphorylated. Phosphofructokinase (EC 2.7.1.11) was assayed by a modification of the method Of UNDERWOOD AND NEWSHOLME 2°. The assay medium contained in a volume of 1.8 ml: 50 mM Tris-HC1 (pH 7.5), 5 mM MgCI~, o.2 mM NADH, 4 mM glucose 6-phosphate, 2 mM AMP, 200 mM KC1, 0.3 mM KCN with 3-9 I.U. of phosphohexose isomerase (EC 5.3.I.9) , 0.5 I.U. of aldolase (EC 4.I.2.7), IO I.U. of triosephosphate isomerase (EC 5.3.1.1) and 3.6 I.U. of glycerophosphate dehydrogenase (EC 1.1.1.8). io/*1 of supernatant were added and the reaction started with 0.2 ml of 20 mM ATP, final concentration 2 raM. Pyruvate kinase (EC 2.7.1.4o) was assayed by the method of B~CHER AND PFLEIDERER21. In 1.8 ml the assay medium contained: 50 mM Tris-HC1 (pH 7.5), 8 mM MgSO4, 75 mM KC1, 2 mM ADP, 0. 3 mM N A D H and 18 I.U. of rabbit muscle lactate dehydrogenase (EC 1.1.1.27). 2/*1 of the supernatant were added and the reaction started with 0.2 ml of 30 mM phosphoenolpyruvate. In all cases, the change in absorbance was followed at 34 ° n m in an Unicam SP 800 spectrophotometer at 3 o°, using the automatic cell changer. When all three enzymes were assayed on the same sample of supernatant, the order of assay was glucokinase, phosphofructokinase and pyruvate kinase. A unit of enzyme activity is defined as that amount that will convert I /*mole of substrate per rain under the conditions described. The supernatant protein concentration was determind by the method of LowRY et al. 22 using a bovine serum albumin standard. Plasma glucose was assayed b y the method of HUGGET AND NIXON23 and plasma nonesterified f a t t y acid by the method of DOLE AND MEINERTZ24. .A, modification of Method C of HALES AND RANDLE25,26 was used for determination of the plasma insulin concentration. 3o/*1 of plasma or standard ox insulin were mixed with 50/*1 of precipitated antibody and incubated for 24 h at 4 °. 0.2 ml of I125I~insulin (1.25 ng/ml) was then added and the reaction stopped after 16 h at 4 ° by filtration on cellulose acetate membranes (Oxo, Oxoid Division, London). The radioactivity of the precipitate was measured by scintillation counting. Chemicals

ATP, NADP +, NADH, Glc-6-P, Phosphoenolpyruvate, ADP and all enzymes used in the assays were obtained from C. F. Boehringer and Soehne, London. Glucose oxidase, peroxidase and hydrocortisone were obtained from the Sigma Chemical Co. L-Thyroxine was obtained from British Drug Houses. Growth hormone (bovine) was prepared b y MR. B. SLATER (ref. 27). The bovine serum albumin standard was Fraction V from the Armour Pharmaceutical Co., Eastbourne, Sussex. All other chemicals were the best grade obtainable from British Drug Houses, Hopkin and Williams or May and Baker. Biochim. Biophys. Acta, 184 (1969) 287-298

g

7

GLUCOSE,

INSULIN

AND

NONESTERIFIED

FATTY

ACID

CONCENTRATIONS

FROM HYPOPHYSECTOMIZED

RATS

6

8

Sham-operated

P a ir- f ed

1.82 4- o . i o

2-40 ~ o.15

2.30 J: 0.2o

1.95 ~: o.13

0.75 ± 0.05*

i.o

28.2 4- 1. 5

31.3 ± 2.4

33 .0 4- 1.5

27-1 i

9.8 ~- I.O*

13.5 ± 0.9

24.0 ± 1.2

17.o ± 0.9

21. 5 ± 1.8

3.9 :~: 0.22*

5.60 -k 0.75

9.65 ± 0.79

IO.Oi ± o.81

9.45 4- 0.7o

2.70 4_ 0.23*

Units/,oo g body wt.

* S i g n i f i c a n t l y different from all c o n t r o l r e s u l t s b y S t u d e n t ' s t t e s t ; P < o.ooi.

4

20

36

Units/ whole liver

125 i

9

121 ± 2

142 ~- 5

133 ± 4

114 ± 3

Glucose (mg/Ioo ml)

Units/g supernatant protein

n

Units/g liver

Plasma

Glucokinase activity

Weight

Age

Controls :

Hypophysectomizedrats

Group

7 ° ~: 9

58 ~ 12

i o o ± 12

90 4- 8

33 ~- 6*

Insulin (l~units/ml)

AND

4-

I0"

443 ± 20

222

Nonesterified fatty acid (l~M)

The results are from d a t a pooled from e x p e r i m e n t s p e r f o r m e d on s e ve ra l days. On each d a y age c o n t r o l a n i m a l s were k i l l e d w i t h t h e o t h e r groups.

GLUCOKINASE ACTIVITY AND PLASMA DIFFERENT CONTROL GROUPS

TABLE I

>

m ~q

>

©

(/?

O

tO

GLUCOKINASE ACTIVITY AFTER HYPOPHYSECTOMY

29I

RESULTS

Table I shows a comparison of glucokinase activity of hypophysectomized animals with different control animals; age, sham-operated, weight and pair-fed controls. The activity was lower in the hypophysectomized animals than in all of the control groups. The range of the activity in the hypophysectomized group was o.35-1.2 units/g liver and 6.o-15.3 units/g supernatant protein. The activity of the control group showed some variation. However, in each experiment age controls were killed with the other control groups and no significant difference (P < 0.05) between the enzyme activity of the two control groups was observed whether activity was expressed as units/g liver or units/g supernatant protein. Differences were observed between the activity of pair-fed and age controls when activity was expressed per liver or per ioo g body weight due to a drop in mean liver weight form io to 7 g over the period of food restriction. TABLE II THE ACTIVITY OF HEKOKINASE AFTER HYPOPHYSECTOMY T h e a s s a y u s e d w a s t h a t d e s c r i b e d i n MATERIALS AND METHODS, w i t h r e p l a c e m e n t of IOO m M glucose b y 0. 5 mM glucose.

Group

Age c o n t r o l Pair-fed control Hypophysectomized

Hexohinase activity Units/g liver

Units/g supernatant protein

0.36 ± 0.03

4.5 + 0.4

0.44 ± 0.05 0.42 ~z 0.03

5.2 ± 0.5 5.1 ± 0.3

Since no correction has been made for the contribution of the hexokinases in the assay, the activity of hexokinase after hypophysectomy was measured (Table II). The activity did not change after the treatment. The mean plasma insulin concentration of the hypophysectomized group was consistently lower than any of the control groups. The variation in the plasma insulin values in the control groups was only apparent. In any one experiment, no significant difference between the age and other control group was seen, although the actual control values obtained varied from experiment to experiment. In a large series of experiments performed on hypophysectomized rats, it was noted that every animal that was judged (by the criteria of slow weight gain and only partially atrophied adrenals) to be incompletely hypophysectomized had normal plasma insulin and glucokinase activity. A fall in the plasma insulin level seemed to be a good indication of complete hypophysectomy. Phosphofructokinase and pyruvate kinase activities did not behave in parallel after hypophysectomy. P y r u v a t e kinase activity from hypophysectomized rats was lower than the activity in either age or weight controls, no matter how the activity is expressed (Table I I I ) . Phosphofructokinase showed a fall only when liver weight was used in the calculation of activity. When expressed as units/g liver or as units/g supern a t a n t protein the activity did not change. Biochim. Biophys. Acta, 184 (1969) 2 8 7 - 2 9 8

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C . M . DAWSON, C. N. HALES

TABLE III A COMPARISON OF THE EFFECTS OF HYPOPHYSECTOMY PHOSPHOFRUCTOKINASE

W I T H A G E A N D W E I G H T C O N T R O L A N I M A L S ON

AND PYRUVATE KINASE ACTIVITIES

T h e number of rats in each group was 4. T h e experimental group was compared with the control group. Group

E n z y m e activity Units/g liver

Units/g

Units~whole

Units/zoo g

supernatant protein

liver

body wt.

Phosphofructokinase Age control Hypophysectomy

2.32 :j_ 0.09 2.44 ~ 0.04

26.6 :k 0.7 26. 7 ~: 0.5

19.5 ± 0.6 11.9 :j_ 1.1"*

8.51 ~: 0.23 8.22 ± 0.29

P y r u v a t e kinase Age control Hypophysectomy

73.8 ~ 6.0 36.9 ± lO.7"

846 ± 7 ° 427 -G IOO**

613 ± 41 184 i 65**

268 ~-: 65 92 ± 9*

Phosphofructokinase Weight control Hypophysectomy

2.13 ± 0.08 2.o6 ± o.o8

29.05 ~__ 1.16 27-oo z~: o.45

18.56 --4=_0.78 lO.42 ~ o.72

P y r u v a t e kinase Weight control Hypophysectomy

66.1 ~ 4.2 33.0 ± 2.1

895 ~ 45 433 • 34

772 ~_ 14 166 :t: 14

11.43 ~ o.41 6.88 ± o.32

478 ± 12 lO 7 ± 6***

*.P < 0.0 5 . **P < o.oi. * * * P < o.ooi.

Thyroxine administration. The effect of injection of normal and hypophysectomized rats with thyroxine (IOO/~g/ioo g body weight) twice daily for 3 days has been studied. Plasma insulin in normal animals was unaffected by the treatment, as was glucokinase activity except for a slight rise when activity was expressed per g protein. In hypophysectomized rats, thyroxine had no effect on the low glucokinase activity. However, the plasma glucose and insulin in the hypophysectomized rats were depressed by the treatment. Growth hormone Injection of bovine growth hormone (2oo ~g/rat) into hypophysectomized rats for 12 or 48 h had no effect on either glucokinase activity or plasma insulin concentration. The lack of effect on the plasma insulin is in agreement with the result of RANDLE AND Y O U N G 14.

Hydrocortisone The effect on glucokinase activity, plasma insulin and glucose of hydrocortisone injection (2 mg/Ioo g rat) into hypophysectomized rats is shown in Table V. 6 h after the commencement of injections, no change was observed in any of the parameters measured. At 12 h the plasma insulin value was raised but the glucokinase activity was unaltered by the treatment. With longer times of treatment the plasma insulin rose still further, followed by a rise in plasma glucose. After 48 h of treatment, glucokinase activity increased by 33 % over the untreated hypophysectomized animal level. Biochim. Biophys. Acta, 184 (1969) 287-298

~D oo

.,q

~o

t~

0.88 z~ o . I o

I . i i 4- o.18 1.42 + o.18

1.59 4- o.12"

Hypophysectomized + hydrocortisone (I2 h)

Hypophysectomized

Hypophysectomized + hydrocortisone for 24 h

H y p o p h y s e c t o m i z e d 4- h y d r o c o r t i s o n e for 48 h

*P < 0.05 . ** P < 0.02. ***P < o.oi. § P < o.ooi .

1.63 4- 0.07*

0.86 4- o.16

Hypophysectomized + hydrocortisone (6 h)

Hypophyzectomized + hydrocortisone for 72 h

0.94 4- o.18

24.2 4- 1.6"

25.8 ± 2.1"

22.2 4- 2. 4

15. 4 4- 2. 7

12.2 4- 0.9

12.1 + 1. 7

13.1 4- 2.7

9.4 4- 0.8**

9.9 4- 1.7"

7.5 ± 0.9

5.1 ± I.O

4.2 ± 0. 4

4.3 4- 1. 3

4.3 ~- i . I

151 4- 4***

146 4- 8*

128 ± 4

128 4- 2

121 ± 7

124 4- 4

127 4- 2

Glucose (rag/zoo ml)

Units/whole liver

Units/g liver

Units/g supernatant protein

Plasma

Glucokinase activity

Hypophysectomized

Group

129 4- 4 °*

159 4- 48*

lO4 4- 13"

46 4- 5

92 4- 13"*

5 ° 4- 5

45 ± 5

Insulin (l~units/ml)

-

752 -~ 90§

474 ± 49***

243 4- 14

169 4- 29

-

--

--

Nonesterified fatty acid (l~M)

The n u m b e r of r a t s in each g r o u p was 4. R a t s were i n j e c t e d w i t h 2 m g h y d r o c o r t i s o n e p e r I oo g r a t a t zero t i m e a n d t h e r e a f t e r a t 12-h i n t e r v a l s . T r e a t ed a n i m a l s were c o m p a r e d w i t h u n t r e a t e d .

EFFECT OF INJECTION OF HYDROCORTISONE INTO HYPOPHYSECTOMIZED RATS FOR 6, 12, 24, 48 AND 72 h

TABLE IV

~0 %3 O3

,..4

>

~Z >

©

C. M. DAWSON,

294

Biochim.

Biophys.

Acta,

184 (1969)

287-298

C. N. HALES

GLUCOKINASE ACTIVITY AFTER HYPOPHYSECTOMY

295

Hydrocortisone with growth hormone 6 h after the injection of hydrocortisone (2 mg/ioo g rat) with growth hormone (200 ~g/rat) the plasma insulin concentration was raised whereas the injection of the two hormones separately at the same dosage had no effect. After 12 h of administration of both hormones together the glucokinase activity was slightly above the untreated value. Longer term treatment with both hormones together (Table V) had a more marked effect on glucokinase activity, a significant difference being observed after 24 h of the treatment•

Hydrocortisone ÷ ,growth hormone 60C o

]

40C

2oc o o

o

~

Hydrocortisone 400f

g •

0

6 ~ ~ T,me of treatment (days)

Fig. I. T h e effect of t r e a t m e n t of h y p o p h y s e c t o m i z e d a n i m a l s w i t h h y d r o c o r t i s o n e w i t h a n d w i t h o u t g r o w t h h o r m o n e for different t i m e s on p l a s m a i n s u l i n (Zl) a n d liver g l u c o k i n a s e a c t i v i t y , e x p r e s s e d as u n i t s / g s u p e r n a t a n t p r o t e i n ( • ) . T h e d a t a are t a k e n f r o m t h e tables a n d are c o m p i l e d b y e x p r e s s i n g t h e v a l u e s as a p e r c e n t a g e of t h e u n t r e a t e d h y p o p h y s e c t o m i z e d v a l u e of t h e exp e r i m e n t . T h e b a r s i n d i c a t e t h e s t a n d a r d error of t h e m e a n .

Fig. I summarizes the effects on glucokinase activity and plasma insulin of the administration to hypophysectomized rats of hydrocortisone with and without growth hormone. The results were compiled by expressing the values at each of the times after the start of the injections as a percentage of the untreated hypophysectomized value. The rise in plasma insulin was greater and appeared slightly earlier with both hormones together than with hydrocortisone alone. DISCUSSION

These results show that after hypophysectomy rat liver glucokinase activity is depressed. One problem in assessing alterations in enzyme activity after hypophysectomy is the validity of the control animals used. The daily food intake of hypophysecBiochim. Biophys. Acta, 184 (1969) 2 8 7 - 2 9 8

290

C. M. DAWSON, C. N. HALES

tomized animals is lower than normal, and starvation itself leads to an alteration of the activity of m a n y enzymes including glucokinase. In these experiments the fall in glucokinase activity was apparent when hypophysectomized animals were compared with age, weight, sham-operated and pair-fed control animals. The fall cannot be accounted for by a fall in the activity of the hexokinase isoenzymes. These findings agree with those of BORREBAEKet al. 's but are at variance with those of RENOLD et al. 17 and SHARMA et al. 16. In the early experiments of RENOLD et al. 17 the glucose, utilization of liver slices was estimated by measuring the removal of [14C~glucose from the incubating medium with time. No account was taken of the contribution of glucose-6-phosphatase to the results obtained. Glucose-6-phosphatase activity is reduced in livers from hypophysectomized rats 2s,29. A fall in the activity of this enzyme might therefore have masked a reduction of glucose utilization. SHARMA e! al. 16 assayed glucokinase activity in liver extracts under optimum conditions and found an impaired response of fasted hypophysectomized rats to refeeding but found that in fed animals the activity was in the normal range. However, no details were given concerning the time of the operation relative to the experiment. Hypophysectomy produces a complex deficiency state 3°, it is therefore difficult to ascertain the possible causes for the lowered glucokinase activity encountered here. Plasma insulin and glucose have been assigned roles in the regulation of glucokinase activityT,,1 13. Since the plasma glucose level in these animals differs little from the control value, it seems unlikely that glucose plays a part in altering the enzyme activity. It seems possible, therefore, that the low plasma insulin of hypophysectomized animals m a y be a cause of the low glucokinase activity seen here. Consistent with this explanation was the fact that in all instances a rise in plasma insulin preceded the rise in enzyme activity. In comparison with treatment with hydrocortisone alone, following combined treatment with hydrocortisone and growth hormone there was an earlier rise in the plasma insulin concentration and this was associated with an earlier rise in glucokinase activity. We have unsuccessfully attempted to investigate the role of insulin in the reduction of glucokinase activity in hypophysectomized rats by injecting these animals with insulin. There was severe hypoglycaemia 6 h after as little as 5 ° munits insulin per animal. The rise in plasma insulin concentration in hypophysectomized rats after hydrocortisone administration could be a result of increased glucose production by the liver, presumably by gluconeogenesis, since liver glycogen is low after hypophysectomy 31, causing increased release of insulin from the pancreas. Glucokinase activity of hypophysectomized animals treated with hydrocortisone did not reach the control level. A possible explanation of this is that after 3 days of steroid treatment the animals are beginning to develop steroid diabetes (shown by the rise in the plasma glucose and nonesterified fatty acid concentration). It has been shown that administration of hydrocortisone, with or without growth hormone, results in a lowering of glucokinase activity 32. The mechanism of the changes in activity observed here have been further investigated by a study of the kinetics of the activity of the crude supernatant and partially purified enzyme (separated from the hexokinases) under different conditions (C. M. DAWSON unpublished observations). Following hypophysectomy, no change has been observed in the K m glucose of the partially purified enzyme. This finding contrasts with that in similar studies of the partially purified enzyme from steroid diabetic animals when the K m glucose of the enzyme was raised. The normal kinetics of the Biochim. Biophys. Acta, 184 (1969) 287-298

GLUCOKINASE ACTIVITY AFTER HYPOPHYSECTOMY

297

partially purified enzyme form hypophysectomized animals is consistent with the reduction of enzyme activity being caused by a fall in enzyme concentration. Unlike the conditions of starvation and alloxan diabetes 33-35, hypophysectomy does not result in a lowering of all three key glycolytic enzymes; phosphofructokin ase activity does not change after this treatment. A small drop in phosphofructokinase activity has been reported in hypophysectomized rats a4. Administration of growth hormone and hydrocortisone to normal animals for 4 days also results in no change of phosphofructokinase activity but a lowering of glucokinase and pyruvate kinase activities 32. It seems that the "functional genic unit" concept as applied to rat liver glucokinase, phosphofructokinase and pyruvate kinase is not supported by these studies. Our studies have shown that the regulation of glucokinase is complex. The mechanism of changes in the activities of the three enzymes requires extensive investigation before conclusions can be drawn about the relative rates of synthesis. ACKN OWLEDGEMENTS

We are indebted to Professor F. G. Young for his interest and encouragement, and to Miss M. Thomas for performing the hypophysectomies. This work was supported by the Medical Research Council of Great Britain and the British Diabetic Association. REFERENCES i 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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c . M . DAWSON, C. N, HALES

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A. F. C. C. G. G.

Biochim. Biophys. Acta, 184 (1969) 287-298