- Email: [email protected]
Hormonal and dietary regulation of hepatic glucokinase
HORMONAL
AND DIETARY REGULATION HEPATIC GLUCOKINASE
OF
CHAKRAVARTHI SHARMA, RAJANI MANJESHWAR and SIDNEY WEINHOUSE The Fels Research Institute, Temple University Medical School, Philadelphia, Pennsylvania THE wide scope of regulatory activity displayed by the liver can be appreciated by examining the contents of the previous symposium, conducted here one year ago; the variety of subject matter of the present symposium makes it clear that interest in this subject has not waned since that time. Our role will be to report work now being carried out in our laboratory on some of the factors which control the phosphorylation of glucose in this organ. Perhaps no other single enzyme would be expected to play a wider role in the functional activities of liver than one that initially activates glucose, a substance which serves not only as a metabolic fuel, but also as a major source of chemical building blocks for the multitudinous variety of synthetic processes occurring in this organ. Despite the importance attributable to this enzyme, until recently very little definitive information was available concerning its properties. A large number of biochemical and physiological studies, with experimental systems ranging in organizational complexity from the liver slice to the intact animal, has pointed to the glucose phosphorylation system as a rate controlling step in the metabolism of glucose in this organ;(r-4) studies carried out particularly with liver slices by Ashmore, Cahill and their collaborators,(s) and by Chaikoff and coworkers,(*) utilizing the isotope tracer technique, have provided information on approximate liver glucose phosphorylation rates under a variety of dietary and hormonal conditions. This is summarized in Table 1, taken from data in a review by Cahill and collaborators.(l) This table shows that glucose phosphorylation is high in the normally fed rat liver slices and is relatively unaffected by cortisone or insulin treatment, or by adrenalectomy or hypophysectomy. It is, however, lowered approximately 50 per cent by fasting. In the diabetic animal there is a marked alteration in the pattern of glucose phosphorylation. In the untreated diabetic, glucose utilization is low and is not affected by adrenalectomy, hypophysectomy or cortisone The experimental work of the authors was supported by grants from the American Cancer Society and the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. 189
190
C. SHARMA, R. MANJESHWAR AND S. WEINHOUSE
treatment. Only insulin administration restores glucose phosphorylation to normal. These findings led us, several years ago, to a study of the isolated hepatic phosphorylation system. Fortunately, the enzyme is in the soluble portion of the cytoplasm, is reasonably stable, and lends itself well to reliable estimation of its activity by spectrophotometric assay of the product, glucose-6-phosphate. Our findings, as we previously reported at last year’s symposium,(s) led us to conclude that the hepatic glucokinase system is indeed rate-limiting for glucose utilization in conditions of glucose and insulin deprivation, and led us to explore further into the factors which regulate this enzyme. Perhaps the most interesting property of this enzyme is that it has a high Michaelis constant for glucose; approximately 1 x 10-2~ as compared with reported values of 10-4~or lower for other hexokinases. This high Km, which requires a glucose concentration of about 200 mg per 100 ml for a half-maximal phosphorylation rate, admirably explains why the liver cell, with a membrane highly permeable to glucose transport, only takes up glucose from the blood when the level is high. TABLE
1
Endocrine Effects on Rat Liver Glucose Utilization I Condition
Glucokinase activity
Normal,
untreated cortisone insulin adrenalectomized hypophysectomized fasted
high high high high high low
Diabetic,
untreated insulin adrenalectomized hypophysectomized adrenalectomized + cortisone
low high low low low
While these properties of the hepatic enzyme system were being studied, a host of additional studies by other investigators have extended these findings and provided much new information. Among these are papers by Vaughan and co-workers,(7) by Ilyin and Shanygina,@) by Lange and Kohn,cQ) by Walker et al.,(lOJl) by Niemeyer et a1.,(12J3) by Sols and collaborators,(14) and by Blumethal et al.(ls) Amongst the most significant of these recent reports are those of Sols and collaborators,(l4) and of Walker,(ll) demonstrating the presence in rat liver not only of the responsive glucose-ATP phosphotransferase, but in addition, a constitutive hexokinase,
HORMONAL
AND
DIETARY
which apparently does not alterations. We are grateful results before publication. existence of this additional of both enzymes, and we low Km enzyme hexokinase Dietary
REGULATION
OF HEPATIC
GLUCOKINASE
191
change appreciably with dietary and hormonal to Dr. Sols who informed us of these interesting When we received word from Dr. Sols of the enzyme, we revised our studies to include assays have followed Sols’ terminology by calling the and the high Km enzyme glucokinase.
Efects
The data shown in Table 2 summarize dietary effects on these enzymes. Columns 2 and 3 show that glucokinase is high in the fed animal, and is lowered to about 40 per cent of its initial value after 72 hr fasting. Under the same conditions essentially no change occurs in the hexokinase level. TABLE 2 Effects of Fasting and Refeeding with’Sugars on Hepatic Glucose-ATP Phosphotransferases of Normal Rats Rats were fasted 3 days and then were given 5 mmoles of the designated sugar in 3 ml of water by stomach tube. After 4 hr the animals were killed for assay of hepatic phosphotransferases. Each value is the mean f the average deviation of separate assays on three animals.
Condition of rat
Fed Fasted 24 hr 48 hr 12 hr Refed glucose fructose galactose mannose
Units/g liver Hexokinase 0.16+0.01
Units/total
Glucokinase
Hexokinase
1.23kO.12
1.3OkO.32
I I
liver
Units/100 g body wt.
Glucokinase
Hexokinase
Glucokinase
10.29k1.9
0.5lkO.14
4.OlkO.69
0.18+0.02 1.02+0.10 1.46kO.20 0.13+0.02 0.47*0.01 0.92+0.08 0.19 f 0.02 0.45 f 0.05 1.14kO.12
8.27kO.57 3.38kO.30 2.7OkO.30
0.50+0.07 0.37 +0.03 0.43 +0.03
2.87kO.17 1.39kO.07 1.04+0.10
0.16+0.03 1.06+0.08 0.15 +O.Ol 0.72kO.07 0.14*0.01 0.68kO.02 0.13+0.03 0.69kO.04
8.09kO.59 5.13kO.39 4.53 +0.37 4.34kO.23
0.48 kO.09 0.44+0.01 0.37+0.01 0.3320.09
3.19kO.21 2.05kO.25 1.18+0.10 1.88kO.13
1.24kO.24 1.10~0.03 0.94kO.08 0.85+0.22
The responsiveness of the glucokinase to glucose is impressively demonstrated by the data on the 5th line of this table, showing that in as little as 4 hr after glucose feeding its activity is back to the normal range. The specificity of the enzyme to glucose is also indicated by the observation that similar feeding of fructose, galactose or mannose does not restore glucokinase activity as rapidly as does glucose. In all instances there is no change in hexokinase activity. The data in the two columns on the right show essentially the same quantitative degree of responsiveness when activities are calculated on the basis of total liver. To determine whether or
192
C. SHARMA,
R.
MANJESHWAR
AND
S. WEINHOUSE
not the effects of fasting and refeeding represent changes in the content of enzyme protein the effects of glucose feeding were tested in the presence of puromycin and ethionine, both of which are specific inhibitors of protein synthesis(r6J7) Lines one and three of Table 3 demonstrate that in 4 hr after glucose feeding to fasted rats glucokinase is restored to normal. If the fasted animal is given an injection of puromycin, no change in activity TABLE 3 Effect of Puromycin on Hepatic ATP-Glucose Phosphotransferases Fasted animals had food withheld 3 days. They were then given either glucose as in Table 1, or an intraperitoneal injection of puromycin in a dosage of 3.5 mg per 100 g, or both. Rats were killed after 4 hr for phosphotransferase assay. Each value is the mean f the average deviation of 3 separate assays
Units/g liver Condition and treatment Fasted Fasted plus puromycin Fed Fed plus puromycin Refed Refed plus puromycin
Hexokinase 0.15 +0.02
I
Glucokinase
Units/total Hexokinase
0.62 + 0.02 0.14 + 0.03
liver Ghlcokinase
Units/100 g Hexokinase
3.21 kO.09 0.4OM.05
Glucokinase 2.10+0.08
0.16 + 0.03 0.65 + 0.09 0.12 f 0.05 3.43kO.10 0.02 f 0.04 1.23 +0.08 1.32kO.12 10.36kO.13
0.43kO.02 0.64kO.03
2.12+0.03 4.18f0.11
0.19 kO.04 0.15+0.02
0.69kO.05 0.40+0.02
4.98kO.08 3.52kO.20
0.4920.07
2.19kO.07
1.32 f 0.08 1.53 + 0.06 11.86k0.32 5.8020.30 1.10+0.10 0.64 +0.04
0.15 + 0.02 D.68 +0.06
0.78 kO.08
3.48kO.33
occurs. However, if the puromycin is injected at the time glucose is fed, glucokinase activity does not increase. Again, there were no changes in hexokinase activity in these experiments. The same response to puromycin is obtained if the activities are calculated on the basis of total liver. Table 4 shows similar data for ethionine-treated animals. Here again, the restoration in enzyme activity which occurs on refeeding does not occur when ethionine is simultaneously administered. The specific effect of ethionine is demonstrated in the last line, which shows that the inhibition of enzyme activity is reversed by additional administration of methionine. These data lead us to assume that the restoration in glucokinase activity which occurs in 4 hr following glucose feeding is due to the synthesis of new enzyme protein. Effects of Insulin on Glucokinase
A remarkable effect of insulin in restoring the hepatic glucokinase activity of alloxan-diabetic rats was reported in preliminary form at last year’s liver symposium. These findings are further documented by studies typified by
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
193
TABLE 4 Effect of Ethionine on Hepatic ATP-Glucose Phosphotransferases Three-day fasted rats were fed 5 mmoles of glucose in 3 ml of water by stomach tube; either alone, or simultaneously with an intraperitoneal injection of 50 mg ethionine; or with 50 mg ethionine and 100 mg methionine. Each value is the mean F the average deviation of three separate assays
Units/g liver Conditions and treatment Fasted Refed Refed + ethionine Refed + ethionine and methionine
Hexokinase
Glucokinase
Units/total Hexokinase
liver
Units/ 100 g rat
Glucokinase
Hexokinase
Glucokinase
0.18+0.03 0.45 + 0.06 0.93kO.02 0.19 f 0.02 1.24f 0.08 0.98f0.03
3.53kO.21 6.15+0.10
0.58kO.02 0.53f0.04
2.01+0.08 3.52kOO.5
0.17f0.02
0.55rtO.12
0.98kO.19
3.1750.60
0.5OzbO.01 1.64f0.32
0.16+0.01
0.97 kO.01 0.96kO.10
5.96kO.40
0.51 kO.01
3.18kO.74
the data shown in Fig. 1. Here there is shown the time course of hexokinase and glucokinase activities in alloxan-diabetic rats, as influenced by administration and withdrawal of insulin. Alloxan-diabetic rats were given a series of insulin injections over a period of 3 days, then insulin was withdrawn and enzyme activities measured over the following 5 days. The line made by the open circles clearly shows that insulin has little or no effect on the hexokinase. The line made by the shaded circles indicated that within 4 hr following the injection of insulin, the blood glucose begins to fall and reaches essentially normal values in 24 hr where it remains as long as insulin is given. Insulin was withdrawn at 72 hr, as indicated by the vertical arrow. Twenty-four hours later, the blood glucose was still at a low level, but then rose rapidly and reached the typical diabetic level of over 400 mg per 100 ml in 48 hr. The glucokinase levels are designated by the triangles. Values are characteristically low, at about 0.3 units per g, in the alloxan-diabetic rat, but rose rapidly beginning about 10 hr after insulin injection, to reach essentially the normal level by 24 hr where it remains as long as insulin is given. Following withdrawal of insulin, the glucokinase level remained high for 48 hr, but then gradually dropped to the low diabetic level by about 5 days where it remains. It is interesting to note the sharp dissociation in time between the effects of insulin withdrawal on blood glucose level and on liver glucokinase. In 48 hr after insulin withdrawal, the blood glucose was near maximal, whereas at this time there was no appreciable lowering of hepatic glucokinase. Clearly, these are separate manifestations of the hormone.
194
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
2.4
,600 600
2.0
500
2
1.6
400
8 5 I
e
300
5 g
1.2 0.6
E
0.4
200 100
0” uy o1 5 m
4
1624.46
72 ON
24
46
INSULIN
72 OFF
66
no
FIG. 1 Effect of insulin on hepatic glucose-ATP phosphotransferases in alloxan diabetic rats. Alloxan diabetic rats excreting 7-12 g of glucose per day in the urine were given a single dose of glucagon-free insulin, 5 units/100 g body weight, and on the second and third days were given the same dosage of protamine zinc insulin. The arrow indicates the time of the last insulin injection at 72 hr. Blood glucose levels are the shaded circles, with values given in the right ordinate. Hexokinase activities are given by the open circles,and glucokinase activities by the triangles, with values in the left ordinate. Each point is the mean of three values and the ranges are given by the vertical lines through the points.
Other Hormones
In view of the well-established effects of the adrenal steroids on enzyme adaptation in liver,os) we examined the effects of adrenalectomy and of cortisone administration. We already mentioned earlier that adrenalectomy or cortisone had relatively little effect on glucose utilization by liver slices. The data shown in Table 5 indicate further that neither hexokinase nor TABLE
5
Effects of Fasting and Refeeding on Glucose-ATT’ Phosphotransferases Adrenalectomized Rats Average of 3 separate assays Activity units/g liver Condition Fed Fasted 24 hr 48 hr Refed 4 hr 24 hr
Activity units/total liver
Hexokinase
Glucokinase
Hexokinase
Glucokinase
0.25 f 0.04
0.86 f 0.04
2.53 kO.08
6.96 +0.72
0.28 f 0.03 0.17 kO.05
0.96 f 0.06 0.32+_ 0.02
2.04&0.11 1.06+_0.12
6.82kO.31 1.96kO.12
0.21 f 0.06 0.21+0.01
0.36kO.04 0.75 +0.06
1.48 f 0.46 1.51 kO.22
2.48 + 0.46 5.53 +0.48
in
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
195
glucokinase is markedly influenced by the adrenal cortex. The “fed” glucokinase level was somewhat lower than normal in the adrenalectomized rats, but the activity dropped in the normal fashion on fasting. The recovery of activity in response to glucose refeeding was somewhat more sluggish in the adrenalectomized than in normal animals, but the glucokinase level was restored to the normal range in 24 hr. Because recent studies have pointed to a stimulating effect of thyroid hormone on protein synthesis,(ls) it was interesting to learn whether the thyroid gland is necessary for responsiveness to glucose. The data in Table 6 TABLE 6 Effects of Fasting and Refeeding on Glucose-ATP Thyroidectomized Rats
Phosphotransferases
in
Averages of three separate assays Units/g liver Condition
Hexokinase
Units/total
liver
Glucokinase
Hexokinase
Glucokinase
0.15 kO.03
1.07kO.14
0.68kO.19
4.80k0.49
0.14&-0.02 0.23 + 0.02
1.12+0.20 1.06kO.03
0.43 f 0.07 0.58 L-O.08
3.43 f. 0.42 2.74kO.33
0.22 f 0.05 0.19rf:o.o3
1.00+0.06 1.09~0.10
0.51 f 0.03 1.09kO.33
2.32 kO.64 5.59 f 0.97
I Fed Fasted 24 hr 48 hr Refed 4 hr 24 hr
are somewhat difficult to interpret. Fasting or glucose refeeding did not alter the specific activity of either phosphotransferase. On the basis of total liver weight, however, the glucokinase level dropped about 40 per cent after a 48 hr fast and, although no recovery was noted in 4 hr, as is regularly observed in normal rats, the enzyme activity was back to normal in 24 hr. Thus the thyroid gland also does not seem to exert any pronounced action on hepatic glucose utilization. Table 7 shows similar data obtained for hypophysectomized rats. Unfortunately these studies have not yet been completed. In keeping with other data, pointing to an impairment of adaptive response of liver enzymes in hypophysectomized animals,(aO) we found (a) that glucokinase activity was quite low in 24 hr of fasting as compared with essentially no change in normal rats; and (b) in 4 hr after glucose feeding, there was no recovery of glucokinase activity. As usual, there were no significant changes in hexokinase activity. These experiments need further amplification, particularly to see how rapidly glucokinase activity is regained after glucose feeding.
196
C. SHARMA,
R.
MANJESHWAR
AND
S. WEINHOUSE
TABLE 7 Effects of Fasting and Refeeding on Glucose-ATP Phosphotransferases Hypophysectomized Rats Averages of three separate assays
I dition
Units/g liver
Units/total
in
liver
--
Glucokinase
Hexokinase
Glucokinase
0.17+0.02
1.59kO.12
1.16+0.04
10.95 kO.66
0.14+0.05
0.95 +0.08
1.03 * 0.54
6.88 + 1.32
0.21* 0.04
0.44+0.06
0.99 + 0.26
2.09 + 0.26
0.16+0.04
0.35 + 0.06
0.91 kO.16
1.85kO.64
0.19 kO.05
0.412 0.05
0.89kO.12
1.92 + 0.42
Hexokinase _-
Fed Fasted 18hr Fasted 24 hr Fasted 48 hr Fasted, Refed 4 hr I
.
Hepatic Glucokinase in the Frog
Because of the pronounced effects of fasting on liver glucokinase activity, it was of especial interest to determine the activities of these enzymes in the livers of hibernating animals, who would have received no dietary carbohydrate intake for long periods. Frogs for laboratory investigation are usually stored in a state of hibernation in the refrigerator, sometimes for many months. We found the liver hexokinase activity to be considerably higher in such animals, about 2-24 times that of the rat (Table 8). Glucokinase, however, was just about as high in these animals as in normal, carbohydrate-fed rats. When such hibernating frogs were warmed up to TABLE
Glucose-ATP
8
phosphotmnsferases in Frog Single Values Units/g liver
Fasting, 4 days at 25°C
(Rana pipiend
Units/total
Condition Hibernating at 5°C
Liver
liver
Hexokinase
Glucokinase
Hexokinase
Glucokinase
0.41 0.41
0.97 0.89
0.53 0.62
1.26 1.34
0.46 0.46
0.85 1.12
0.55 0.48
1.02 1.17
-
HORMONAL AND DIETARY REGULATION
OF HEPATIC GLUCOKINASE
197
room temperature, even without feeding, there was again no change in either enzyme activity. Apparently the frog does not exhibit the same adaptation in glucokinase activity as the warm blooded animals. It would be interesting to extend this study to some hibernating mammal. Location of Phosphotransferasesin the Liver Cell
The presence of two glucose-ATP phosphotransferases in liver raises the question whether these enzymes are present in different cell types. Some data obtained by us recently suggest that the glucokinase may be in parenchymal cells, which make up the preponderance of liver cells, and the hexokinase may be in the bile duct cells, which normally comprise about 4 per cent of the total.t21) When a liver carcinogen, such as 3’-methyldimethylaminoazobenzene (3’-methyl DAB), is fed to rats there is an early proliferation of bile duct cells and a corresponding decrease in parenchymal cells; by about 3-4 weeks the duct cells might comprise as much as 50 per cent of the total. If the aforementioned distribution of the enzymes prevails,. there should occur at this time a corresponding increase in hexokinase and a decrease in glucokinase. A group of young male rats was placed on an 18 per cent casein diet containing 0.06 per cent of 3’-methyl DAB, and a control group was given the same diet without the carcinogen. At weekly intervals two carcinogen-fed rats and one control rat were killed and the enzymes were assayed. This procedure was carried out for 11 weeks, by which time tumors are beginning to develop. The results, though not highly striking, are nevertheless clear-cut. All of the control rats, as shown on the left hand bar (Fig. 2) throughout the experimental period, had normal 1.6 1.6I I
'y$'3
•OLUCOKINA~E/
4 6 6 7 WEEKS ON 3’-Me
6 DAB
9 IO DIET
FIG. 2 Effect of feeding 3’-methyl DAB on rat liver hexokinase and glucokinase. Shaded bars give hexokinase, clear bars give glucokinase, and vertical lines indicate ranges of two values. Control diet bar at extreme left is the average of single values for control animals fed the diet without the carcinogen over the entire 1 l-week period. r4
198
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
levels of glucokinase and hexokinase. Beginning at the 4th week, there ensued a marked elevation in the hexokinase, and a corresponding reduction in the glucokinase, and this condition persisted throughout the experimental period of 11 weeks. Samples of all livers assayed in this experiment were examined histologically by Andrew J. Donnelly of the Institute for Cancer Research, to whom we are greatly indebted. In general the correlation between the degree of bile duct proliferation and the increase in ratio of hexokinase to glucokinase was excellent. Whenever the level of hexokinase was high, the histologic report revealed marked duct cell hyperplasia, and whenever the hexokinase was normal, the histologic report revealed little or no bile duct hyperplasia. The data are regarded as being in accord with the presumption that the hexokinase is present in the bile duct epithelium, whereas the glucokinase may be the sole or preponderant enzyme of the parenchymal cells. We recognize, however, that this presumed correlation may be fortuitous, since at best, histologic study, applied to the focalized nests of bile duct proliferation scattered in irregular fashion throughout the liver can give only rough approximations of the true proportions of various liver cell types. Other means of altering the liver cell population are now being investigated to try to shed more light on the question of the localization of the hepatic glucose phosphotransferases. DISCUSSION
The data leave no doubt that glucose and insulin are crucial factors in hepatic glucokinase induction. Their respective functions in this process deserve some comment. The rapid recovery in glucokinase activity on glucose feeding following carbohydrate deprivation points to a powerful substrate induction comparable to that observed with hepatic tryptophan pyrrolase.@a) However, the situation is quite different from that involved in tryptophan pyrrolase induction in that there appears to be little if any effect of the cortical steroids on this process. Cahill, Ashmore and their collaborators previously indicated there was little effect of adrenalectomy or cortisone administration on glucose utilization in liver slices,(l) and the present results support their findings by demonstrating a similar lack of effect on liver glucokinase. In contrast with tryptophan pyrrolase, in which the intact, functioning adrenal gland or replacement therapy is required for substrate induction, glucose induction of glucokinase activity seems to be independent of the adrenals. Preliminary data indicate that the pituitary and thyroid glands also play no role in the induction of this enzyme. However, it is clear that insulin is necessary for substrate induction. It is interesting to note that an effect of insulin in stimulating hexokinase activity was suggested 16 years ago by the studies of Price, Colowick and Cori.(a3) The present results confirm such an effect of insulin, but additionally make
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
199
clear that (a) the effect is restricted to liver; (b) the effect is on the synthesis of the enzyme rather than a direct in vitro stimulation. Although this effect of insulin is not immediate, and does not occur in vitro, it is nonetheless one of the most striking of the biochemical effects of this hormone. If glucose were the only requirement for hepatic glucokinase activity, it would be difficult to understand why the enzyme should not be present during fasting, since glucose is always present in the blood in this condition, and it should be particularly high in the liver, where glucogenesis proceeds at maximal rates during fasting. In view of the pronounced effects of insulin in restoring the glucokinase activity in the liver of the alloxan-diabetic rat, it might be argued in fact that glucose itself might act indirectly in the induction of the enzyme by stimulating insulin secretion in the same manner as cr-methyltryptophan induces tryptophan pyrrolase activity by stimulating the adrenals.@r) Supporting this suggestion is the fact that insulin would not be secreted during fasting, despite the presence of glucose in the liver, but would be secreted during hyperglycemia; that is under precisely those conditions which lead to the enhancement of glucokinase activity. Another question remaining for clarification is the mechanism of these inductive effects. Studies designed to differentiate between various possibilities are now under way. The fact that the effects of insulin treatment and withdrawal in the alloxan-diabetic rat develop only after relatively long periods suggests that insulin plays some part in maintaining the structure of the liver cell in a condition which is required for responsiveness to glucose. Krahl has reported considerable evidence for an action of insulin in stimulating hepatic protein synthesis.(25) Although not all hepatic enzymes require insulin for their adaptive synthesis, since some remain unchanged and some even increase in alloxan-diabetes,(ss) it would appear that one of these proteins which is synthesized under the influence of insulin is glucokinase. Whether other enzymes require insulin for adaptive synthesis, is an interesting question for future study. REFERENCES I. 2.
G. F. CAHILL, JR., J. ASHMORE, A. E. RENOLD and A. B. HASTINGS, Blood and the liver, Am. .I. Med. 26. 264-282 (1959). R. LEVINE and I. B. FRITZ, The relation of insulin to liver metabolism,
glucose Diabetes
5, 209-222 (1956). 3. S. WEINHOUSE, 4. 5.
6.
B. FIUEDMANN and G. A. REICHARD, JR., Effects of insulin on hepatic glucose production and utilization, Diabetes 12, l-7 (1963). S. P. CHERNICK, I. L. CHAIKOFF and S. ABRAHAM, Localization of initial block in glucose metabolism in diabetic liver slices, J. Biol. Chem. 193, 793-802 (1951). R. G. SPIRO, J. ASHMORE and A. B. I&STINGS, Studies on carbohydrate metabolism in liver slices, XII. Sequence of events following acute insulin deprivation, J. Biol. Chem. 230, 761-771 (1958). S. WEINHOUSE, V. CIUSTOFALO, C. SHARMA and H. P. MORRIS, in G. Weber (Editor), fva;;~ in Enzyme Regulation, Vol. 1, Pergamon Press, New York, 1963, pp. .
200 I. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
D. A. VAUGHAN, J. P. HANNON and L. N. VAUGHAN, Effects of diet on selected glycolytic enzymes of the rat, Am. J. Pbysiol. 199, 1041-1044 (1960). V. S. ILYIN and K. L. SHANYGMA, On hormonal control of the hexokinase reaction in liver, Problems in Med. Chem., USSR 6, 288-291 (1960). C. G. LANGE and P. KOHN, Substrate specificity of hexokinases, J. Biol. Chem. 236, l-5 (1961). D. G. WALKER, The development of hepatic hexokinases after birth, Biochem. J. 84, 118 P (1962). D. G. WALKER and S. RAo, Some factors affecting the hepatic hexokinases, Biochem. J. 88, 17 P (1963). H. NIEMEYER, L. CLARK-TURRI, E. GARCES and F. E. VERGARA, Selective response of liver enzymes to the administration of different diets after fasting, Arch. Biochem. and Biophys. 98, 77-85 (1962). H. NIE&ER, i. CLARK-T&RI and E. RABAJILLE, Induction of glucokinase by elucose in rat liver. Nature 198. 1096-1097 (1963). E. VIRLIELA, M. SALAS and A. SOLS, Glucokinase ind hexokinase in liver in relation to glycogen synthesis, J. Biol. Gem. PC1 175 (1963). M. D. BLUMENTHAL, S. ABRAHAM and I. L. CHAIKOFF, Arch. Biochem. and Biophys. (in press). A. M. NEMETH and G. DE LA HABA, The effect of puromycin on the development and adaptive formation of tryptophan pyrrolase, J. Biol. Gem. 237,1190-l 193 (1962). E. FARBER, Advances in Cancer Research (S. Weinhouse and A. Haddow, Editors). Academic Press, New York, 7, 387-393 (1963). G. WEBER, G. BANERJEE and S. B. BRONSTEIN, Role of enzymes in homeostasis. III. Selective induction of increases in liver enzymes involved in carbohydrate metabolism, J. Biol. Gem. 236, 3106-3111 (1961). J. R. TATA, L. ERNSTER, 0. LINDBERG, E. ARRHEMIUS, S. PEDERSENand R. HEDMAN, The action of thyroid hormones at the cell level, Biochem. J. 86,408-428 (1963). G. WEBER and A. CANTERO, Effect of hypophysectomy on liver enzymes involved in glycogenolysis and in glucogenesis, Am. J. Physiol. 197, 699-701 (1959). R. DAOUST and A. CANTERO, The numerical proportions of cell types in rat liver during carcinogenesis by 4-dimethyl-aminoazobenzene (DAB), Cancer Research 19, 757-762 (1959). P. FEIGEL~ON and 0. GREENGARD, Regulation of liver tryptophan pyrrolase activity, J. Biol. Chem. 237, 1908-1913 (1962). W. H. PRICE, C. F. Cool and S. P. COLOWICK, The effect of anterior pituitary extract and of insulin on the hexokinase reaction,J. Biol. Chem. 160,633-634 (1945). L. GOLDSTEIN, E. J. STELLA and W. E. KNOX, The effect of hydrocortisone on tyrosine-a-ketoglutarate transaminase and tryptophan pyrrolase activities in the isolated perfused rat liver, J. Biol. Chem. 237,~ 1721-1726 (1962). M. E. KRAHL, The Action of Insulin on Cells. Academic Press, New York, 1961, pp. 111-116. W. M. FITCH, R. HILL and I. L. CHAIKOFF, Hepatic glycolytic enzyme activities in the alloxan-diabetic rat: Response to glucose and fructose feeding.-. J. Biol. Chem. 234,2811-2813 (1959). -
AND DIETARY REGULATION HEPATIC GLUCOKINASE
OF
CHAKRAVARTHI SHARMA, RAJANI MANJESHWAR and SIDNEY WEINHOUSE The Fels Research Institute, Temple University Medical School, Philadelphia, Pennsylvania THE wide scope of regulatory activity displayed by the liver can be appreciated by examining the contents of the previous symposium, conducted here one year ago; the variety of subject matter of the present symposium makes it clear that interest in this subject has not waned since that time. Our role will be to report work now being carried out in our laboratory on some of the factors which control the phosphorylation of glucose in this organ. Perhaps no other single enzyme would be expected to play a wider role in the functional activities of liver than one that initially activates glucose, a substance which serves not only as a metabolic fuel, but also as a major source of chemical building blocks for the multitudinous variety of synthetic processes occurring in this organ. Despite the importance attributable to this enzyme, until recently very little definitive information was available concerning its properties. A large number of biochemical and physiological studies, with experimental systems ranging in organizational complexity from the liver slice to the intact animal, has pointed to the glucose phosphorylation system as a rate controlling step in the metabolism of glucose in this organ;(r-4) studies carried out particularly with liver slices by Ashmore, Cahill and their collaborators,(s) and by Chaikoff and coworkers,(*) utilizing the isotope tracer technique, have provided information on approximate liver glucose phosphorylation rates under a variety of dietary and hormonal conditions. This is summarized in Table 1, taken from data in a review by Cahill and collaborators.(l) This table shows that glucose phosphorylation is high in the normally fed rat liver slices and is relatively unaffected by cortisone or insulin treatment, or by adrenalectomy or hypophysectomy. It is, however, lowered approximately 50 per cent by fasting. In the diabetic animal there is a marked alteration in the pattern of glucose phosphorylation. In the untreated diabetic, glucose utilization is low and is not affected by adrenalectomy, hypophysectomy or cortisone The experimental work of the authors was supported by grants from the American Cancer Society and the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. 189
190
C. SHARMA, R. MANJESHWAR AND S. WEINHOUSE
treatment. Only insulin administration restores glucose phosphorylation to normal. These findings led us, several years ago, to a study of the isolated hepatic phosphorylation system. Fortunately, the enzyme is in the soluble portion of the cytoplasm, is reasonably stable, and lends itself well to reliable estimation of its activity by spectrophotometric assay of the product, glucose-6-phosphate. Our findings, as we previously reported at last year’s symposium,(s) led us to conclude that the hepatic glucokinase system is indeed rate-limiting for glucose utilization in conditions of glucose and insulin deprivation, and led us to explore further into the factors which regulate this enzyme. Perhaps the most interesting property of this enzyme is that it has a high Michaelis constant for glucose; approximately 1 x 10-2~ as compared with reported values of 10-4~or lower for other hexokinases. This high Km, which requires a glucose concentration of about 200 mg per 100 ml for a half-maximal phosphorylation rate, admirably explains why the liver cell, with a membrane highly permeable to glucose transport, only takes up glucose from the blood when the level is high. TABLE
1
Endocrine Effects on Rat Liver Glucose Utilization I Condition
Glucokinase activity
Normal,
untreated cortisone insulin adrenalectomized hypophysectomized fasted
high high high high high low
Diabetic,
untreated insulin adrenalectomized hypophysectomized adrenalectomized + cortisone
low high low low low
While these properties of the hepatic enzyme system were being studied, a host of additional studies by other investigators have extended these findings and provided much new information. Among these are papers by Vaughan and co-workers,(7) by Ilyin and Shanygina,@) by Lange and Kohn,cQ) by Walker et al.,(lOJl) by Niemeyer et a1.,(12J3) by Sols and collaborators,(14) and by Blumethal et al.(ls) Amongst the most significant of these recent reports are those of Sols and collaborators,(l4) and of Walker,(ll) demonstrating the presence in rat liver not only of the responsive glucose-ATP phosphotransferase, but in addition, a constitutive hexokinase,
HORMONAL
AND
DIETARY
which apparently does not alterations. We are grateful results before publication. existence of this additional of both enzymes, and we low Km enzyme hexokinase Dietary
REGULATION
OF HEPATIC
GLUCOKINASE
191
change appreciably with dietary and hormonal to Dr. Sols who informed us of these interesting When we received word from Dr. Sols of the enzyme, we revised our studies to include assays have followed Sols’ terminology by calling the and the high Km enzyme glucokinase.
Efects
The data shown in Table 2 summarize dietary effects on these enzymes. Columns 2 and 3 show that glucokinase is high in the fed animal, and is lowered to about 40 per cent of its initial value after 72 hr fasting. Under the same conditions essentially no change occurs in the hexokinase level. TABLE 2 Effects of Fasting and Refeeding with’Sugars on Hepatic Glucose-ATP Phosphotransferases of Normal Rats Rats were fasted 3 days and then were given 5 mmoles of the designated sugar in 3 ml of water by stomach tube. After 4 hr the animals were killed for assay of hepatic phosphotransferases. Each value is the mean f the average deviation of separate assays on three animals.
Condition of rat
Fed Fasted 24 hr 48 hr 12 hr Refed glucose fructose galactose mannose
Units/g liver Hexokinase 0.16+0.01
Units/total
Glucokinase
Hexokinase
1.23kO.12
1.3OkO.32
I I
liver
Units/100 g body wt.
Glucokinase
Hexokinase
Glucokinase
10.29k1.9
0.5lkO.14
4.OlkO.69
0.18+0.02 1.02+0.10 1.46kO.20 0.13+0.02 0.47*0.01 0.92+0.08 0.19 f 0.02 0.45 f 0.05 1.14kO.12
8.27kO.57 3.38kO.30 2.7OkO.30
0.50+0.07 0.37 +0.03 0.43 +0.03
2.87kO.17 1.39kO.07 1.04+0.10
0.16+0.03 1.06+0.08 0.15 +O.Ol 0.72kO.07 0.14*0.01 0.68kO.02 0.13+0.03 0.69kO.04
8.09kO.59 5.13kO.39 4.53 +0.37 4.34kO.23
0.48 kO.09 0.44+0.01 0.37+0.01 0.3320.09
3.19kO.21 2.05kO.25 1.18+0.10 1.88kO.13
1.24kO.24 1.10~0.03 0.94kO.08 0.85+0.22
The responsiveness of the glucokinase to glucose is impressively demonstrated by the data on the 5th line of this table, showing that in as little as 4 hr after glucose feeding its activity is back to the normal range. The specificity of the enzyme to glucose is also indicated by the observation that similar feeding of fructose, galactose or mannose does not restore glucokinase activity as rapidly as does glucose. In all instances there is no change in hexokinase activity. The data in the two columns on the right show essentially the same quantitative degree of responsiveness when activities are calculated on the basis of total liver. To determine whether or
192
C. SHARMA,
R.
MANJESHWAR
AND
S. WEINHOUSE
not the effects of fasting and refeeding represent changes in the content of enzyme protein the effects of glucose feeding were tested in the presence of puromycin and ethionine, both of which are specific inhibitors of protein synthesis(r6J7) Lines one and three of Table 3 demonstrate that in 4 hr after glucose feeding to fasted rats glucokinase is restored to normal. If the fasted animal is given an injection of puromycin, no change in activity TABLE 3 Effect of Puromycin on Hepatic ATP-Glucose Phosphotransferases Fasted animals had food withheld 3 days. They were then given either glucose as in Table 1, or an intraperitoneal injection of puromycin in a dosage of 3.5 mg per 100 g, or both. Rats were killed after 4 hr for phosphotransferase assay. Each value is the mean f the average deviation of 3 separate assays
Units/g liver Condition and treatment Fasted Fasted plus puromycin Fed Fed plus puromycin Refed Refed plus puromycin
Hexokinase 0.15 +0.02
I
Glucokinase
Units/total Hexokinase
0.62 + 0.02 0.14 + 0.03
liver Ghlcokinase
Units/100 g Hexokinase
3.21 kO.09 0.4OM.05
Glucokinase 2.10+0.08
0.16 + 0.03 0.65 + 0.09 0.12 f 0.05 3.43kO.10 0.02 f 0.04 1.23 +0.08 1.32kO.12 10.36kO.13
0.43kO.02 0.64kO.03
2.12+0.03 4.18f0.11
0.19 kO.04 0.15+0.02
0.69kO.05 0.40+0.02
4.98kO.08 3.52kO.20
0.4920.07
2.19kO.07
1.32 f 0.08 1.53 + 0.06 11.86k0.32 5.8020.30 1.10+0.10 0.64 +0.04
0.15 + 0.02 D.68 +0.06
0.78 kO.08
3.48kO.33
occurs. However, if the puromycin is injected at the time glucose is fed, glucokinase activity does not increase. Again, there were no changes in hexokinase activity in these experiments. The same response to puromycin is obtained if the activities are calculated on the basis of total liver. Table 4 shows similar data for ethionine-treated animals. Here again, the restoration in enzyme activity which occurs on refeeding does not occur when ethionine is simultaneously administered. The specific effect of ethionine is demonstrated in the last line, which shows that the inhibition of enzyme activity is reversed by additional administration of methionine. These data lead us to assume that the restoration in glucokinase activity which occurs in 4 hr following glucose feeding is due to the synthesis of new enzyme protein. Effects of Insulin on Glucokinase
A remarkable effect of insulin in restoring the hepatic glucokinase activity of alloxan-diabetic rats was reported in preliminary form at last year’s liver symposium. These findings are further documented by studies typified by
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
193
TABLE 4 Effect of Ethionine on Hepatic ATP-Glucose Phosphotransferases Three-day fasted rats were fed 5 mmoles of glucose in 3 ml of water by stomach tube; either alone, or simultaneously with an intraperitoneal injection of 50 mg ethionine; or with 50 mg ethionine and 100 mg methionine. Each value is the mean F the average deviation of three separate assays
Units/g liver Conditions and treatment Fasted Refed Refed + ethionine Refed + ethionine and methionine
Hexokinase
Glucokinase
Units/total Hexokinase
liver
Units/ 100 g rat
Glucokinase
Hexokinase
Glucokinase
0.18+0.03 0.45 + 0.06 0.93kO.02 0.19 f 0.02 1.24f 0.08 0.98f0.03
3.53kO.21 6.15+0.10
0.58kO.02 0.53f0.04
2.01+0.08 3.52kOO.5
0.17f0.02
0.55rtO.12
0.98kO.19
3.1750.60
0.5OzbO.01 1.64f0.32
0.16+0.01
0.97 kO.01 0.96kO.10
5.96kO.40
0.51 kO.01
3.18kO.74
the data shown in Fig. 1. Here there is shown the time course of hexokinase and glucokinase activities in alloxan-diabetic rats, as influenced by administration and withdrawal of insulin. Alloxan-diabetic rats were given a series of insulin injections over a period of 3 days, then insulin was withdrawn and enzyme activities measured over the following 5 days. The line made by the open circles clearly shows that insulin has little or no effect on the hexokinase. The line made by the shaded circles indicated that within 4 hr following the injection of insulin, the blood glucose begins to fall and reaches essentially normal values in 24 hr where it remains as long as insulin is given. Insulin was withdrawn at 72 hr, as indicated by the vertical arrow. Twenty-four hours later, the blood glucose was still at a low level, but then rose rapidly and reached the typical diabetic level of over 400 mg per 100 ml in 48 hr. The glucokinase levels are designated by the triangles. Values are characteristically low, at about 0.3 units per g, in the alloxan-diabetic rat, but rose rapidly beginning about 10 hr after insulin injection, to reach essentially the normal level by 24 hr where it remains as long as insulin is given. Following withdrawal of insulin, the glucokinase level remained high for 48 hr, but then gradually dropped to the low diabetic level by about 5 days where it remains. It is interesting to note the sharp dissociation in time between the effects of insulin withdrawal on blood glucose level and on liver glucokinase. In 48 hr after insulin withdrawal, the blood glucose was near maximal, whereas at this time there was no appreciable lowering of hepatic glucokinase. Clearly, these are separate manifestations of the hormone.
194
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
2.4
,600 600
2.0
500
2
1.6
400
8 5 I
e
300
5 g
1.2 0.6
E
0.4
200 100
0” uy o1 5 m
4
1624.46
72 ON
24
46
INSULIN
72 OFF
66
no
FIG. 1 Effect of insulin on hepatic glucose-ATP phosphotransferases in alloxan diabetic rats. Alloxan diabetic rats excreting 7-12 g of glucose per day in the urine were given a single dose of glucagon-free insulin, 5 units/100 g body weight, and on the second and third days were given the same dosage of protamine zinc insulin. The arrow indicates the time of the last insulin injection at 72 hr. Blood glucose levels are the shaded circles, with values given in the right ordinate. Hexokinase activities are given by the open circles,and glucokinase activities by the triangles, with values in the left ordinate. Each point is the mean of three values and the ranges are given by the vertical lines through the points.
Other Hormones
In view of the well-established effects of the adrenal steroids on enzyme adaptation in liver,os) we examined the effects of adrenalectomy and of cortisone administration. We already mentioned earlier that adrenalectomy or cortisone had relatively little effect on glucose utilization by liver slices. The data shown in Table 5 indicate further that neither hexokinase nor TABLE
5
Effects of Fasting and Refeeding on Glucose-ATT’ Phosphotransferases Adrenalectomized Rats Average of 3 separate assays Activity units/g liver Condition Fed Fasted 24 hr 48 hr Refed 4 hr 24 hr
Activity units/total liver
Hexokinase
Glucokinase
Hexokinase
Glucokinase
0.25 f 0.04
0.86 f 0.04
2.53 kO.08
6.96 +0.72
0.28 f 0.03 0.17 kO.05
0.96 f 0.06 0.32+_ 0.02
2.04&0.11 1.06+_0.12
6.82kO.31 1.96kO.12
0.21 f 0.06 0.21+0.01
0.36kO.04 0.75 +0.06
1.48 f 0.46 1.51 kO.22
2.48 + 0.46 5.53 +0.48
in
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
195
glucokinase is markedly influenced by the adrenal cortex. The “fed” glucokinase level was somewhat lower than normal in the adrenalectomized rats, but the activity dropped in the normal fashion on fasting. The recovery of activity in response to glucose refeeding was somewhat more sluggish in the adrenalectomized than in normal animals, but the glucokinase level was restored to the normal range in 24 hr. Because recent studies have pointed to a stimulating effect of thyroid hormone on protein synthesis,(ls) it was interesting to learn whether the thyroid gland is necessary for responsiveness to glucose. The data in Table 6 TABLE 6 Effects of Fasting and Refeeding on Glucose-ATP Thyroidectomized Rats
Phosphotransferases
in
Averages of three separate assays Units/g liver Condition
Hexokinase
Units/total
liver
Glucokinase
Hexokinase
Glucokinase
0.15 kO.03
1.07kO.14
0.68kO.19
4.80k0.49
0.14&-0.02 0.23 + 0.02
1.12+0.20 1.06kO.03
0.43 f 0.07 0.58 L-O.08
3.43 f. 0.42 2.74kO.33
0.22 f 0.05 0.19rf:o.o3
1.00+0.06 1.09~0.10
0.51 f 0.03 1.09kO.33
2.32 kO.64 5.59 f 0.97
I Fed Fasted 24 hr 48 hr Refed 4 hr 24 hr
are somewhat difficult to interpret. Fasting or glucose refeeding did not alter the specific activity of either phosphotransferase. On the basis of total liver weight, however, the glucokinase level dropped about 40 per cent after a 48 hr fast and, although no recovery was noted in 4 hr, as is regularly observed in normal rats, the enzyme activity was back to normal in 24 hr. Thus the thyroid gland also does not seem to exert any pronounced action on hepatic glucose utilization. Table 7 shows similar data obtained for hypophysectomized rats. Unfortunately these studies have not yet been completed. In keeping with other data, pointing to an impairment of adaptive response of liver enzymes in hypophysectomized animals,(aO) we found (a) that glucokinase activity was quite low in 24 hr of fasting as compared with essentially no change in normal rats; and (b) in 4 hr after glucose feeding, there was no recovery of glucokinase activity. As usual, there were no significant changes in hexokinase activity. These experiments need further amplification, particularly to see how rapidly glucokinase activity is regained after glucose feeding.
196
C. SHARMA,
R.
MANJESHWAR
AND
S. WEINHOUSE
TABLE 7 Effects of Fasting and Refeeding on Glucose-ATP Phosphotransferases Hypophysectomized Rats Averages of three separate assays
I dition
Units/g liver
Units/total
in
liver
--
Glucokinase
Hexokinase
Glucokinase
0.17+0.02
1.59kO.12
1.16+0.04
10.95 kO.66
0.14+0.05
0.95 +0.08
1.03 * 0.54
6.88 + 1.32
0.21* 0.04
0.44+0.06
0.99 + 0.26
2.09 + 0.26
0.16+0.04
0.35 + 0.06
0.91 kO.16
1.85kO.64
0.19 kO.05
0.412 0.05
0.89kO.12
1.92 + 0.42
Hexokinase _-
Fed Fasted 18hr Fasted 24 hr Fasted 48 hr Fasted, Refed 4 hr I
.
Hepatic Glucokinase in the Frog
Because of the pronounced effects of fasting on liver glucokinase activity, it was of especial interest to determine the activities of these enzymes in the livers of hibernating animals, who would have received no dietary carbohydrate intake for long periods. Frogs for laboratory investigation are usually stored in a state of hibernation in the refrigerator, sometimes for many months. We found the liver hexokinase activity to be considerably higher in such animals, about 2-24 times that of the rat (Table 8). Glucokinase, however, was just about as high in these animals as in normal, carbohydrate-fed rats. When such hibernating frogs were warmed up to TABLE
Glucose-ATP
8
phosphotmnsferases in Frog Single Values Units/g liver
Fasting, 4 days at 25°C
(Rana pipiend
Units/total
Condition Hibernating at 5°C
Liver
liver
Hexokinase
Glucokinase
Hexokinase
Glucokinase
0.41 0.41
0.97 0.89
0.53 0.62
1.26 1.34
0.46 0.46
0.85 1.12
0.55 0.48
1.02 1.17
-
HORMONAL AND DIETARY REGULATION
OF HEPATIC GLUCOKINASE
197
room temperature, even without feeding, there was again no change in either enzyme activity. Apparently the frog does not exhibit the same adaptation in glucokinase activity as the warm blooded animals. It would be interesting to extend this study to some hibernating mammal. Location of Phosphotransferasesin the Liver Cell
The presence of two glucose-ATP phosphotransferases in liver raises the question whether these enzymes are present in different cell types. Some data obtained by us recently suggest that the glucokinase may be in parenchymal cells, which make up the preponderance of liver cells, and the hexokinase may be in the bile duct cells, which normally comprise about 4 per cent of the total.t21) When a liver carcinogen, such as 3’-methyldimethylaminoazobenzene (3’-methyl DAB), is fed to rats there is an early proliferation of bile duct cells and a corresponding decrease in parenchymal cells; by about 3-4 weeks the duct cells might comprise as much as 50 per cent of the total. If the aforementioned distribution of the enzymes prevails,. there should occur at this time a corresponding increase in hexokinase and a decrease in glucokinase. A group of young male rats was placed on an 18 per cent casein diet containing 0.06 per cent of 3’-methyl DAB, and a control group was given the same diet without the carcinogen. At weekly intervals two carcinogen-fed rats and one control rat were killed and the enzymes were assayed. This procedure was carried out for 11 weeks, by which time tumors are beginning to develop. The results, though not highly striking, are nevertheless clear-cut. All of the control rats, as shown on the left hand bar (Fig. 2) throughout the experimental period, had normal 1.6 1.6I I
'y$'3
•OLUCOKINA~E/
4 6 6 7 WEEKS ON 3’-Me
6 DAB
9 IO DIET
FIG. 2 Effect of feeding 3’-methyl DAB on rat liver hexokinase and glucokinase. Shaded bars give hexokinase, clear bars give glucokinase, and vertical lines indicate ranges of two values. Control diet bar at extreme left is the average of single values for control animals fed the diet without the carcinogen over the entire 1 l-week period. r4
198
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
levels of glucokinase and hexokinase. Beginning at the 4th week, there ensued a marked elevation in the hexokinase, and a corresponding reduction in the glucokinase, and this condition persisted throughout the experimental period of 11 weeks. Samples of all livers assayed in this experiment were examined histologically by Andrew J. Donnelly of the Institute for Cancer Research, to whom we are greatly indebted. In general the correlation between the degree of bile duct proliferation and the increase in ratio of hexokinase to glucokinase was excellent. Whenever the level of hexokinase was high, the histologic report revealed marked duct cell hyperplasia, and whenever the hexokinase was normal, the histologic report revealed little or no bile duct hyperplasia. The data are regarded as being in accord with the presumption that the hexokinase is present in the bile duct epithelium, whereas the glucokinase may be the sole or preponderant enzyme of the parenchymal cells. We recognize, however, that this presumed correlation may be fortuitous, since at best, histologic study, applied to the focalized nests of bile duct proliferation scattered in irregular fashion throughout the liver can give only rough approximations of the true proportions of various liver cell types. Other means of altering the liver cell population are now being investigated to try to shed more light on the question of the localization of the hepatic glucose phosphotransferases. DISCUSSION
The data leave no doubt that glucose and insulin are crucial factors in hepatic glucokinase induction. Their respective functions in this process deserve some comment. The rapid recovery in glucokinase activity on glucose feeding following carbohydrate deprivation points to a powerful substrate induction comparable to that observed with hepatic tryptophan pyrrolase.@a) However, the situation is quite different from that involved in tryptophan pyrrolase induction in that there appears to be little if any effect of the cortical steroids on this process. Cahill, Ashmore and their collaborators previously indicated there was little effect of adrenalectomy or cortisone administration on glucose utilization in liver slices,(l) and the present results support their findings by demonstrating a similar lack of effect on liver glucokinase. In contrast with tryptophan pyrrolase, in which the intact, functioning adrenal gland or replacement therapy is required for substrate induction, glucose induction of glucokinase activity seems to be independent of the adrenals. Preliminary data indicate that the pituitary and thyroid glands also play no role in the induction of this enzyme. However, it is clear that insulin is necessary for substrate induction. It is interesting to note that an effect of insulin in stimulating hexokinase activity was suggested 16 years ago by the studies of Price, Colowick and Cori.(a3) The present results confirm such an effect of insulin, but additionally make
HORMONAL
AND
DIETARY
REGULATION
OF HEPATIC
GLUCOKINASE
199
clear that (a) the effect is restricted to liver; (b) the effect is on the synthesis of the enzyme rather than a direct in vitro stimulation. Although this effect of insulin is not immediate, and does not occur in vitro, it is nonetheless one of the most striking of the biochemical effects of this hormone. If glucose were the only requirement for hepatic glucokinase activity, it would be difficult to understand why the enzyme should not be present during fasting, since glucose is always present in the blood in this condition, and it should be particularly high in the liver, where glucogenesis proceeds at maximal rates during fasting. In view of the pronounced effects of insulin in restoring the glucokinase activity in the liver of the alloxan-diabetic rat, it might be argued in fact that glucose itself might act indirectly in the induction of the enzyme by stimulating insulin secretion in the same manner as cr-methyltryptophan induces tryptophan pyrrolase activity by stimulating the adrenals.@r) Supporting this suggestion is the fact that insulin would not be secreted during fasting, despite the presence of glucose in the liver, but would be secreted during hyperglycemia; that is under precisely those conditions which lead to the enhancement of glucokinase activity. Another question remaining for clarification is the mechanism of these inductive effects. Studies designed to differentiate between various possibilities are now under way. The fact that the effects of insulin treatment and withdrawal in the alloxan-diabetic rat develop only after relatively long periods suggests that insulin plays some part in maintaining the structure of the liver cell in a condition which is required for responsiveness to glucose. Krahl has reported considerable evidence for an action of insulin in stimulating hepatic protein synthesis.(25) Although not all hepatic enzymes require insulin for their adaptive synthesis, since some remain unchanged and some even increase in alloxan-diabetes,(ss) it would appear that one of these proteins which is synthesized under the influence of insulin is glucokinase. Whether other enzymes require insulin for adaptive synthesis, is an interesting question for future study. REFERENCES I. 2.
G. F. CAHILL, JR., J. ASHMORE, A. E. RENOLD and A. B. HASTINGS, Blood and the liver, Am. .I. Med. 26. 264-282 (1959). R. LEVINE and I. B. FRITZ, The relation of insulin to liver metabolism,
glucose Diabetes
5, 209-222 (1956). 3. S. WEINHOUSE, 4. 5.
6.
B. FIUEDMANN and G. A. REICHARD, JR., Effects of insulin on hepatic glucose production and utilization, Diabetes 12, l-7 (1963). S. P. CHERNICK, I. L. CHAIKOFF and S. ABRAHAM, Localization of initial block in glucose metabolism in diabetic liver slices, J. Biol. Chem. 193, 793-802 (1951). R. G. SPIRO, J. ASHMORE and A. B. I&STINGS, Studies on carbohydrate metabolism in liver slices, XII. Sequence of events following acute insulin deprivation, J. Biol. Chem. 230, 761-771 (1958). S. WEINHOUSE, V. CIUSTOFALO, C. SHARMA and H. P. MORRIS, in G. Weber (Editor), fva;;~ in Enzyme Regulation, Vol. 1, Pergamon Press, New York, 1963, pp. .
200 I. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
C. SHARMA,
R. MANJESHWAR
AND
S. WEINHOUSE
D. A. VAUGHAN, J. P. HANNON and L. N. VAUGHAN, Effects of diet on selected glycolytic enzymes of the rat, Am. J. Pbysiol. 199, 1041-1044 (1960). V. S. ILYIN and K. L. SHANYGMA, On hormonal control of the hexokinase reaction in liver, Problems in Med. Chem., USSR 6, 288-291 (1960). C. G. LANGE and P. KOHN, Substrate specificity of hexokinases, J. Biol. Chem. 236, l-5 (1961). D. G. WALKER, The development of hepatic hexokinases after birth, Biochem. J. 84, 118 P (1962). D. G. WALKER and S. RAo, Some factors affecting the hepatic hexokinases, Biochem. J. 88, 17 P (1963). H. NIEMEYER, L. CLARK-TURRI, E. GARCES and F. E. VERGARA, Selective response of liver enzymes to the administration of different diets after fasting, Arch. Biochem. and Biophys. 98, 77-85 (1962). H. NIE&ER, i. CLARK-T&RI and E. RABAJILLE, Induction of glucokinase by elucose in rat liver. Nature 198. 1096-1097 (1963). E. VIRLIELA, M. SALAS and A. SOLS, Glucokinase ind hexokinase in liver in relation to glycogen synthesis, J. Biol. Gem. PC1 175 (1963). M. D. BLUMENTHAL, S. ABRAHAM and I. L. CHAIKOFF, Arch. Biochem. and Biophys. (in press). A. M. NEMETH and G. DE LA HABA, The effect of puromycin on the development and adaptive formation of tryptophan pyrrolase, J. Biol. Gem. 237,1190-l 193 (1962). E. FARBER, Advances in Cancer Research (S. Weinhouse and A. Haddow, Editors). Academic Press, New York, 7, 387-393 (1963). G. WEBER, G. BANERJEE and S. B. BRONSTEIN, Role of enzymes in homeostasis. III. Selective induction of increases in liver enzymes involved in carbohydrate metabolism, J. Biol. Gem. 236, 3106-3111 (1961). J. R. TATA, L. ERNSTER, 0. LINDBERG, E. ARRHEMIUS, S. PEDERSENand R. HEDMAN, The action of thyroid hormones at the cell level, Biochem. J. 86,408-428 (1963). G. WEBER and A. CANTERO, Effect of hypophysectomy on liver enzymes involved in glycogenolysis and in glucogenesis, Am. J. Physiol. 197, 699-701 (1959). R. DAOUST and A. CANTERO, The numerical proportions of cell types in rat liver during carcinogenesis by 4-dimethyl-aminoazobenzene (DAB), Cancer Research 19, 757-762 (1959). P. FEIGEL~ON and 0. GREENGARD, Regulation of liver tryptophan pyrrolase activity, J. Biol. Chem. 237, 1908-1913 (1962). W. H. PRICE, C. F. Cool and S. P. COLOWICK, The effect of anterior pituitary extract and of insulin on the hexokinase reaction,J. Biol. Chem. 160,633-634 (1945). L. GOLDSTEIN, E. J. STELLA and W. E. KNOX, The effect of hydrocortisone on tyrosine-a-ketoglutarate transaminase and tryptophan pyrrolase activities in the isolated perfused rat liver, J. Biol. Chem. 237,~ 1721-1726 (1962). M. E. KRAHL, The Action of Insulin on Cells. Academic Press, New York, 1961, pp. 111-116. W. M. FITCH, R. HILL and I. L. CHAIKOFF, Hepatic glycolytic enzyme activities in the alloxan-diabetic rat: Response to glucose and fructose feeding.-. J. Biol. Chem. 234,2811-2813 (1959). -