- Email: [email protected]
The role of hormones in glucose repression in rat liver
THE ROLE OF HORMONES IN GLUCOSE REPRESSION IN RAT LIVER* HANS D. S(3LING,~ JOEL KAPLAN,~ MARIE ERBSTOESZER and HENRY C. PITOT§ MeArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wisconsin INTRODUCTION PREVIOUS reports from this laboratory (1, 2) have demonstrated that the forced feeding of enzymatically hydrolyzed casein, or of an equimolar mixture of essential amino acids, to rats that have been fed a protein-depleted diet for five days resulted in dramatic increases in the activities of the enzymes, serine dehydratase and ornithine transaminase. Evidence also has been presented that the dietary-induced increases in enzyme activity can be completely prevented by the simultaneous or delayed administration of glucose or fructose, but not by an isocaloric amount of fat (3). The use of inhibitors of R N A synthesis (actinomycin D and fluoroorotic acid) has shown that the induction of these enzymes is initially inhibited by these compounds, but at later times during the induction, further induction is insensitive or slightly stimulated by these inhibitors (1).
RNA Synthesis and Serine Dehydratase Induction and Repression More recent studies have demonstrated that doses of high energy T radiation of 400 R or greater significantly inhibit the dietary-induced increase in the activity of this enzyme at periods where such increases are also sensitive to the effects of actinomycin D (4). These data together suggest that initially the induction of the enzyme, serine dehydratase, requires concomitant synthesis of messenger R N A but later synthesis becomes resistant to the effects of actinomycin or 7 radiation and, thereafter, does not require concomitant R N A synthesis. In Fig. 1 is seen an experiment designed to determine the period after initiation of enzyme synthesis by a single dose of * This work was supported in part by grants from the National Cancer Institute (CA 07175) and The American Cancer Society (P-314). I"Special Postdoctoral fellow of the North Atlantic Treaty Organization. ~:Postdoctoral fellow of the National Cancer Institute (CA 21666). § Career Development Awardee of the National Cancer Institute (CA-29,405). 171
172
HANS D. S~LING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
casein hydrolysate during which further enzyme synthesis is resistant to the effects of the antibiotic. It should be noticed that enzyme induction remained completely sensitive to the effects of actinomycin for the first hour and onehalf after the initial intubation. Until the eighth to ninth hour after enzyme SDH UNITS 7oc
60C
/.
50C
~oo
I
,..°,...,
/
/ I
300
200
IOC
\
/!
j/"
,
,...." / //L..'" / / / / " ~
""
l
....... D.D. + ACT D -D.D. ONLY
- - - BASAL SINGLE DOSE CURVE
TIME, HOURS
FIo. 1 Actinomycin D (ACT D) sensitivity and resistance of serine dehydratase induction after an initial single dose of I g of caseinhydrolysate. The format of the experiment is described in ref. 5. At each time point noted by white circles, groups of animals were givena second dose (D.D.) of caseinhydrolysate with and without actinomycin.
induction was initiated, further induction of the enzyme is completely resistant to the effects of the antibiotic. After the ninth hour, however, further enzyme induction is again sensitive to actinomycin administration. Similar, if not identical, data have been obtained in our laboratory by the use of 3/ radiation (unpublished observations). During the period of actinomycin D resistance no R N A synthesis is required indicating that genetic translation is the only process occurring during this time period. For the synthesis of any enzyme the period of actinomycin D resistance has been termed "the template lifetime", indicating that during this period one is seeing the expression of a stable messenger R N A template. As indicated in the introduction, studies (2) demonstrated that the early administration of glucose completely prevented the casein hydrolysate-
HORMONES AND GLUCOSE REPRESSION
173
induced levels of serine dehydratase. I n addition, administration of glucose during periods when enzyme synthesis proved resistant to actinomycin also prevented any further increase in the level of the enzyme. Recent studies using quantitative i m m u n o c h e m i c a l techniques (6) have verified these conclusions showing that in fact glucose shuts off the synthesis of serine dehydratase both at periods of actinomycin D resistance as well as actinomycin D sensitivity (Table 1). It can be noted from the data in Table 1 that if TABLE 1 INDUCTIONAND REPRESSIONOF SERINEDEHYDRATASEIN LIVERSOF INTACTRATS* l*C-Valine incorporation No. of animals
Protein control, 0 Amino acids 6 hr 10 hr 18 hr Amino acids + glucose (10 hr) Amino acids (10 hr) + delayed glucose (6 hr only) L-Tryptophan (10 hr) Glucagon (10 hr)
Serine dehydratase activity
Serine dehydratase/g liver
Soluble proteins/g liver
units/g liver 4.4 -4- 0.4
d.p.m. 66.0 -4- 23.0
320,000
60.0 -4- 14.3 125.6 + 27.1 260.0 4- 20.8
836.0 4- 137.0 1816.0 4- 396.0 1850.0 + 427.0
325,000
5.6 -4- 0.5
40.5 4- 24.3
324,000
43.6 4- 13.4 71.9 4- 6.1 509.7 + 57.0
150.1 4- 45.9 1365.0 -4- 60.0 5858.0 4- 706.0
489,000 273,000 465,000
* The experimental details may be found in ref. 6. An incomplete mixture of essential amino acids, lacking valine and methionine, was administered at the times indicated. L-Tryptophan, 0.37 mmole, was administered at 0 time and 6 hr to the group receiving this single amino acid. Glucagon was administered intraperit0neally at a dose of 0.2 mg/100 g body weight at 0 time and again at 6 hr. The hours in parentheses denote the time of death in relation to 0 time, with the exception of the "6 hr only" designation for delayed glucose which signifies the only time of glucose administration. glucose is given at zero time and continued throughout the experimental conditions an abrupt and complete cessation of the enzyme synthesis occurs. If the administration of glucose is delayed until 6 hr after the administration of the initial dose of a m i n o acids there is a m a r k e d inhibition of the rate of enzyme synthesis as measured at 10 hr. If this is c o m p a r e d with the rate of enzyme synthesis either at 6 to 10 hr and corrected for the total incorporation of amino acid into soluble protein, glucose administration has inhibited enzyme synthesis by 9 5 % . Therefore, these experiments demonstrate quite conclusively that a repression of serine dehydratase synthesis
174
HANSD. SOLING,JOELKAPLAN,MARIEERBSTOESZERAND HENRYC. PITOT
mediated by glucose occurs at the level of genetic translation. Furthermore, in Table 1 one may see that there is an actual increase in the rate of enzyme synthesis between the 6- and 10-hr period. At both of these points under the conditions of this experiment enzyme synthesis would be resistant to actinomycin D. It may also be noted from the data of Table 1 that the hormone, glucagon, at a level of 0.2 mg per 100 g of body weight induces a dramatic increase in both activity and radioactivity of serine dehydratase within 10 hr. Previous studies by Peraino and Pitot (2) have demonstrated that glucagon given in doses used in this experiment was as efficient in the induction of serine dehydratase as was the administration of amino acid mixtures. Furthermore, as with induction by amino acids, shortly after the initiation of glucagon administration enzyme synthesis became resistant to the effects of actinomycin D in much the same manner as after amino acid administration. As seen from the data of Table 1 glucagon induces the greatest rate of synthesis of those materials utilized. Although the data are not shown in this Table, glucose will repress glucagon-induced synthesis of serine dehydratase, although not as efficiently as when amino acids are the inducing agents.
Serine Dehydratase Induction in Adrenalectomized Animals Previous studies (1) have shown that the feeding of a 90% casein diet or the administration of casein hydrolysate resulted in significant increases in
TABLE2 INDUCTION AND REPRESSION OF SERINE DEHYDRATASE AND HORMONES IN LIVERS OF ADRENALECTOMIZED RATS*
14C-Valine incorporation No. of animals
Protein (0 %) Amino acid (6 hr) Amino acid + glucose (6 hr) Glucagon (6 hr) Hydrocortisone (6 hr) Fasting (6 hr) Fasting (18 hr)
Enzyme activity
Serine dehydratase/g liver
Soluble proteins/g liver
units/g liver 40.0 4- 15.0 301.0 4- 18.0
196 4- 52 1481 4- 96
d.p.m. 179,000 4- 18,000 241,000 4- 17,000
161.0 4- 9.0 583.0 -4- 24.0
6604-117 27354-205
239,000 ! 19,000 253,000 + 20,000
323.0 + 11.0 139.0 4- 16.0 250.5 -4- 45.9
1944:t:238 9704-151 14424-199
267,000 ± 19,000 197,000 :k 18,000 123,000 4- 15,000
* See legend of Table 1 for details. Hydrocortisone (10 mg) was injected intraperitoneally at 0 time.
HORMONES AND GLUCOSE REPRESSION
175
serine dehydratase activity in the livers of adrenalectomized rats. In addition, cortisone administration effected a marked rise in serine dehydratase levels of rats maintained on a laboratory chow diet over the period of one week. Studies by Rosen and Nichol (7) have also demonstrated that the acute administration of cortisone will lead to dramatic increases in serine dehydratase activity within 24 hr. Fasting of adrenalectomized animals has also been shown to result in a marked rise in serine dehydratase activity in liver (8). As seen from the data of Table 2 amino acid administration induces a significant rise in the rate of synthesis of serine dehydratase in liver. This rise is markedly inhibited by glucose although not as efficiently as seen in the intact a n i m a l Again, the administration of glucagon appears to be the most efficient inducer of serine dehydratase in the liver of the adrenalectomized rat. Hydrocortisone, in confirmation of other studies (7), is seen to induce a significant rise in the rate of synthesis of this enzyme, almost as great as that seen after amino acid administration. Fasting, as has been reported, also increases both enzyme activity and the rate of synthesis.
The Role of Insulin in Glucose Repression in Rat Liver As was reported earlier (2), administration of varying levels of glucose produces an inhibition of serine dehydratase synthesis of varying degrees. Even at a level of 0.5 g of glucose per dose there is a dramatic inhibition of serine dehydratase synthesis. In more recent studies, seen in Fig. 2, it may 3000 e-,.e CH, o - - o C.H. +GLUC. (20rag) CH. ,I- GLUC. (50rag)
25oo
.L
~
c.,. + GLUC.(Jso~Q)
H
C.H. + GLUC.(3OOmg)
J I
/
~/o
~ 2000 1500
1000
50O
0
I 6
I 12
I 17
FIG. 2 The effect of low doses of glucose on the induction of serine dehydratase by casein hydrolysate. The format of the experiment is described in ref. 2, the only difference being the dosages of glucose utilized as may be seen in the figure.
176
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
be noticed that even lower levels of glucose effectively prevent the increase in serine dehydratase reported earlier. As little as 150 nag of glucose given at 6-hr point effectively causes a reduced rate of increase in serine dehydratase activity. At quite low levels of glucose (50 mg), as seen from the graph, there appears to be an initial stimulation of the rate of serine dehydratase increase. Although the differences noted here are not statistically significant, these data are in line with those seen in yeast wherein glucose at low levels (10-4M) actually stimulates the synthesis rather than inhibits that of the inducible enzyme, ct-glucosidase (J.-P., Jost, unpublished results). The actual mechanism for this stimulation by glucose is not immediately apparent although the previous finding reported from this laboratory that administration of glucose actually increases A T P levels in liver suggests that such a mechanism may account for these data. Since the administration of glucose to animals stimulates a dramatic release of insulin from the pancreas, it is reasonable to suggest that this hormone plays a major role in the effect of glucose seen in these experiments. This is especially true since Suda and his associates (9) have demonstrated that serine dehydratase levels in liver of alloxan-diabetic rats are markedly higher than that seen in normal animals. Administration of low protein, high carbohydrate diets did not appear to alter the level of this enzyme in the liver of alloxan-diabetic animals. In order to substantiate and extend Suda's studies, rats were made diabetic by the intravenous administration of alloxan (45 m g / k g male rat) and held for one week. At the end of this time blood sugars were determined and only those animals exhibiting a fasting blood sugar of greater than 300 mg% were used for the experiments reported here. All animals were placed in rooms lighted from 9 p.m. to 9 a.m. and maintained in the dark for the daylight periods. Food was given to the animals at 9 in the morning and removed at 5 in the afternoon. For the forced-feeding studies animals were intubated with casein and other materials at 8-hr intervals beginning at 12:00 noon after an overnight fast. Animals were sacrificed 24 hr after the initial intubation. Insulin was administered at a dose of 2 units every 2 hr the day before sacrifice and once on the day of sacrifice 6 units of Lente insulin were also given the night before. All enzyme analyses were carried out with an automated system that had been previously described (10). Enzyme units are defined as /xmoles of product formed per hour. As seen from the data of Table 3 which includes measurement of the enzymes serine dehydratase and ornithine transaminase induced after maintenance of animals on a 12% protein diet, administration of casein hydrolysate over a 24-hr period results in a marked gtimulation in the level of both enzymes. Furthermore, the administration of glucose markedly inhibits this increase in the case of serine dehydratase and to a lesser extent in the case of ornithine transaminase. However, the administration of casein with and
HORMONES AND GLUCOSE REPRESSION
177
TABLE3 SERINE DEHYDRATASE (SDH) AND ORNrrmNE TRANSAMINASE (OT) LEVELS IN NORMAL AND DIABETIC LIVER*
SDH
Control (12 Yoprotein diet) Control -t- casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin q- glucose
OT
Normal
Diabetic
Normal
Diabetic
4.9 d- 0.8 33.2 4. 4.0 6.3 4. 0.8 5.7 4- 1.0
24.0 q- 3.1 16.6 ± 1.8 20.5 4. 2.0 30.0 4- 4.2 26.5 4- 2.2
1.5 -4- 0.2 7.7 4. 1.5 6.7 4. 0"8 6.6 4. 0.6
6.1 4. 0.6 8.2 4. 0.7 6.2 + 1.0 6.8 4. 0.4 5.7 4. 0.8
16.7 4- 2.4
3.8 q- 0.4
* All animals (8-12 rats per condition) were maintained on a 12~ protein diet as described in the text. Casein hydrolysate (casein) was administered via stomach tube (I g/4 CO with and without glucose or fructose (2 g/dose) every 8 hr as described in the text. See text for insulin dosages. Enzyme activities are expressed as #M product formed/hr/liver x 103 4standard error of the mean. without glucose and insulin or fructose has little or no effect on the level of these enzymes in the diabetic liver. It should be noted that in support of Suda's data the level of serine dehydratase in the liver of an animal maintained on a 12% protein diet is quite high, being almost at the level induced by casein alone. A possible exception to this is seen in the case of the animal given casein, insulin and glucose wherein ornithine transaminase appears to be repressed. Thus, it would appear that the alloxan-diabetic animal maintained under these conditions exhibits very little, if any, effect of insulin a n d / o r glucose and fructose. Since the administration was over a 24-hr period and previous studies have demonstrated that the half-life of serine dehydratase is about 5 hr (6), it would seem that if repression did occur it should easily be discernible under the conditions of these experiments. A question remains as to whether or not these animals are, in fact, diabetic and this was verified on the basis of blood sugar (greater than 300 nag %) as well as the data seen in Table 4. In Table 4 the levels of glucokinase in these animals are demonstrated. As can be seen in the alloxandiabetic animals, the level of glucokinase is quite low. However, when insulin or insulin and glucose is given, there is a dramatic increase in the level of this enzyme. Similar results may be noted from the data of Table 5 wherein the activities of the citrate cleavage enzyme and glucose 6-phosphate dehydrogenase are seen. In this instance, in normal animals the administration of casein plus glucose or fructose results in a significant increase in the level of both of these enzymes. Such an increase is borderline, if there is any at all in the case of the alloxan-diabetic liver. However, when
178
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT TABLE 4 GLUCOKINASE LEVELS IN NORMAL AND DIABETIC LIVER*
Normal 27.9 23.2 34.1 45.7
Control (12 ~ protein diet) Control + casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin + glucose
4- 3.2 4- 1.3 4- 1.2 4- 4.0
Diabetic 3.3 4.7 6.1 7.8 27.8 57.5
4- 1.0 ± 1.8 4- 2.4 4- 1.7 ± 1.9 4- 3.9
* See legend of Table 3 for details. Enzyme activities are expressed as/~M product formed/hr/liver × 102 4- standard error of the mean.
TABLE 5 CITRATE CLEAVAGE ENZYME (CCE) AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE
(G6PD) LEVELSIN NORMALAND DIABETICLIVER* G6PD
CCE
Control (12~ protein diet) Control + casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin + glucose
Normal
Diabetic
18.8 4- 3.1 25.0 q- 2.5 49.8 4- 4.1 42.1 + 3.4
9.8 4- 1.6 8.3 4- 1.0 10.6 4- 1.7 12.4 4- 1.9 44.4 q- 8.4 60.0 4- 6.0
Normal 22.8 46.2 96.8 90.2
± 4.4 4- 5.9 4- 7.4 4- 6.0
Diabetic 9.7 4- 0.8 10.0 4- 1.0 17.9 ± 3.5 20.2 ± 3.3 86.8 4- 15.8 121.54-16.9
* See legend of Table 3 for details. Enzyme activities are expressed as/~Mproduct formed/ hr/liver x 102 4- standard error of the mean. i n s u l i n is given a d r a m a t i c increase occurs in the level of these enzymes in diabetic liver. I n T a b l e 6 is n o t e d the effect of feeding to alloxan-diabetic rats a 90% protein diet together with glucose in the d r i n k i n g water as well as the a d m i n istration of i n s u l i n with a n d without glucose. Perhaps the most striking thing seen with these data is the extremely high level of both serine dehydratase a n d o r n i t h i n e t r a n s a m i n a s e . This m a y be the result of a n extreme s t i m u l a t i o n of gluconeogenesis which is n o r m a l l y present in the diabetic a n d a u g m e n t e d b y the feeding of a high-protein diet. T h e a d m i n i s t r a t i o n of insulin for a 2-day period to these a n i m a l s produces n o significant change in the level of the enzyme, although again, based on half-life studies, if this h o r m o n e h a d been effective changes should be d e m o n s t r a b l e over the time period studied. However, the a d m i n i s t r a t i o n of glucose alone or with i n s u l i n
179
HORMONES AND GLUCOSE REPRESSION TABLE 6 ENZYME LEVELS IN LIVERS OF DIABETIC RATS FED A 90~o PROTEIN DIET* SDH Control 0ontrol + insulin Control + glucose (ad lib) Control + glucose (ad lib) 4- insulin
OT
GK
249.0 4- 21.5 105.7 :k 12.0 2.3 4- 0.5 236.8 4- 26.4 99.3 4- 8.2 30.8 4- 5.7 69.5 4- 26.7
48.0 :k 10.0
6.8 4- 3.5
99.7 4- 14.7
38.1 4- 10.1 37.9 4- 2.9
CCE
G6PD
9.2 4- 0.7 I0.1 4- 1.6 21.9 4- 4.4 24.7 4- 6.0 15.1 4- 1.9
13.1 4- 2.4
37.4 4- 5.4 49.2 4- 11.6
* See text for experimental details. All enzyme activities are expressed as/~M product/hr/ liver × 102 4- standard error of the mean except SDH wherein the factor is 103.
simultaneously with the 90% protein diet does result in significant inhibition of the high levels of both of these enzymes. These results suggest that glucose alone is of much greater significance in the mechanism of glucose repression than is the hormone insulin--at least with respect to these two enzymes. In the case of glucokinase, citrate cleavage enzyme and glucose6-phosphate dehydrogenase, as expected from earlier results, only animals given insulin showed a highly significant increase in the level of these enzymes. As an adjunct to these studies, the effect of diabetes and glucose or fructose with and without insulin on the levels of tryptophan pyrrolase and tyrosine transaminase was ascertained. As seen from the data of Table 7 the TABLE 7 TRYPTOPHAN PYRROLASE (TP) AND TYROSINE a-KETOGLUTARATE TRANSAMINASE (TAKG) LEVELS IN NORMAL AND DIABETIC LIVER*
TP (units/liver) 12 ~ Protein diet Control Control + casein Control + casein Control + casein Control + casein Control + casein
+ + + + +
glucose fructose insulin insulin glucose
90 ~o Protein diet Control Control + insulin Control + glucose (ad lib.) Control + glucose (ad lib.) + insulin
T A K G (units/liver No 2)
Normal Diabetic 22.8 4- 8.4 85.1 4- 6.0 86.3 4- 11.5 121.8 4- 13.1 87.4 + 8.7 148.6 4- 19.5 104.9 4- 24.2 76.4 4- 10.9 125.4 4- 17.6 84.5 4- 9.8 152.4 220.7 120.4 86.9
4444-
15.0 21.6 34.9 13.4
*See legends to Tables 3 and 6 for experimental details. G
Normal 5.5 4- 0.8 17.4 4- 5.9 25.6 4- 5.7 22.0 4- 2.6
Diabetic 9.2 ± 2.4 19.6 4- 3.7 22.3 4- 7.1 11.2 4- 1.2 22.3 4- 4.9 16.4 4- 1.9 53.5 47.6 37.9 18.7
4444-
2.4 1.9 7.5 5.3
180
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
effects of these dietary and hormonal conditions are not readily related to either variable. Administration of casein hydrolysate, as has been earlier demonstrated, results in a dramatic stimulation of the activity of both enzymes. However, glucose or fructose administration has essentially no effect on this in substantiation of earlier results with tyrosine transaminase wherein administration of casein plus carbohydrate actually stimulated the level of this enzyme under certain conditions. In the diabetic liver casein itself appears to cause a significant increase in the level of tyrosine transaminase which can be eliminated by the administration of fructose. Insulin and glucose, however, appear to have little or no effect. Similar results are seen with tryptophan pyrrolase, wherein also insulin plus glucose produces a significant inhibition of the slight increase produced by casein. When feeding a 90% protein diet, only the administration of glucose and insulin produces an inhibition. DISCUSSION
As can be seen from the data of the last five tables, the induction of a number of enzymes is considerably altered by the production of experimental alloxan diabetes. In general, the effects seen may be grouped by enzymes into several groups. Serine dehydratase and ornithine transaminase exhibit little effect of glucose casein or insulin in the liver of the alloxan diabetic animal. Administration of a high-protein diet, however, results in dramatic increases which are prevented by simultaneous glucose administration in the drinking water, but not affected by a 48-hr administration of insulin. This would strongly suggest that the effect of glucose in repressing the synthesis of serine dehydratase is not primarily mediated by insulin, but is probably an extremely complex mechanism which involves several hormones such as glucagon, hydrocortisone, and possibly others. Experiments are now in progress to determine the effect of hypophysectomy in association with alloxan diabetes. If the diabetic animal is adrenalectomized, the levels of serine dehydratase are essentially the same as those seen in the intact diabetic rat. The responses of glucokinase seen in this experiment completely parallel those reported in the literature by a number of other authors (11,12). In the case of the citrate cleavage enzyme and glucose 6-phosphate dehydrogenase the effects are not quite so simple as those seen with glucokinase. Specifically the administration of carbohydrate and protein does result in a significant rise in the level of both of these enzymes. Earlier studies by Ono and Potter (13) had demonstrated that adrenalectomy prevented the fasting-refeeding induction of glucose 6-phosphate dehydrogenase that had been reported by Tepperman (14). In addition, in this latter enzyme there does appear to be an effect of carbohydrate and protein even in the liver of the diabetic animal. A similar, although less significant, increase may be seen in animals fed a 90% protein diet with glucose in their drinking water. Finally, the effects of
HORMONES AND GLUCOSE REPRESSION
181
these various agents on tryptophan pyrrolase and tyrosine transaminase do not appear to fit into any specific pattern. Previous studies had already demonstrated the "anomalous" response of tyrosine transaminase to glucose and adrenal steroids (15). Recent studies by Civen and his associates (16) suggesting that cyclic 3"5"-AMP is important in the regulation of the level of this enzyme are quite significant, particularly on the basis of studies from this laboratory (17) as well as several others. It is possible that this small molecule plays a major role in the changes in enzyme levels that we have described here, but as yet what this role is remains to be elucidated. SUMMARY
Previous studies from this laboratory have demonstrated that the administration of amino acid mixtures to rats previously fed a 0% protein diet will markedly induce the synthesis of several hepatic enzymes. In particular, serine dehydratase is markedly induced under these conditions. The induction is characterized by an initial period of RNA synthesis, but thereafter appears to continue independent of RNA synthesis. That the increases seen are the result of actual changes in the rate of synthesis of the enzyme has been demonstrated by immunochemical methods. Relatively low doses of glucose (150 mg) effectively repress enzyme synthesis under these conditions. Glucagon also induces serine dehydratase, this induction being repressed by glucose. Insulin given to animals partially repressed with glucose has no further effect. However, rats made diabetic with alloxan show marked increases in the level of serine dehydratase, this high level being unaffected by glucose, fructose, or insulin administration. Similar findings may be seen with the mitochondrial enzyme, ornithine transaminase. The enzymes of carbohydrate metabolism, glucokinase, citrate cleavage enzyme and glucose 6-phosphate dehydrogenase, may be induced by protein and glucose in the intact animal, but appear to require glucose and insulin in the diabetic animal. Animals fed a high protein diet show marked increases in serine dehydratase and ornithine transaminase which is not affected by the chronic administration of insulin. These studies demonstrate that glucose repression is a complex phenomenon, probably requiring the interaction of hormones and substrates. The data seen in this paper, however, do not in any way support a primary role for insulin in the mechanism of glucose repression in rat liver. REFERENCES 1. H. C. PITOT and C. PERAINO,Studies on the induction and repression of enzymes in rat liver I. Induction of threonine dehydrase and ornithine-8-transarninase by oral incubation of casein hydrolysate, J. Biol. Chem. 239, 1783-1788 (1964). 2. C. I~RAINO and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver II. Carbohydrate repression of dietary and hormonal induction of threonine dehydrase and ornithine-8-transaminase, J. BioL Chem. 239, 4308-4313 (1964).
182
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
3. H. C. PITOT and C. PE~dNO, Carbohydrate repression of enzyme induction in rat liver, J. Biol. Chem. 238, PC1910-12 (1963). 4. H. C. PiTOT, C. PERAINO.C. LAMARand S. L~HER, The effect of gamma radiation on the dietary and hormonal induction of enzymes in rat liver, Science 150, 901 (1965). 5. H. C. PITOT, C. PERAtNO, C. LAMARand A. L. I~NNAN, Template stability of some enzymes in rat liver and hepatoma, Proc. NatL Acad. Sci. 54, 845-851 (1965). 6. J. P. JOST, E. A. KHAmALLAHand H. C. PITOT, Studies on the induction and repression of enzymes in rat liver V. Regulation of the rate of synthesis and degradation of serine dehydratase by dietary amino acids and glucose, J. Biol. Chem. 243, 3057-3066 (1968). 7. F. ROSEN and C. A. NICHOL, Studies on the nature and specificity of the induction of several adaptive enzymes responsive to cortisol, Advances in Enzyme Regulation 2, 115-135 (1964). 8. H. C. PITOT, Studies on the control of protein synthesis in normal and neoplastic rat liver, P h . D . Dissertation, Tulane University, (1959). 9. E. ISHIKAWA,P. }.~IINIGAWAand M. SODA, Hormonal and dietary control of serine dehydratase in rat liver, J. Biochem. (Japan) 57, 506-513 (1965). 10. H. C. PITOT, N. WRAr'rEN and M. POmIER, The automated assay of complete enzyme reaction rates II. Digital readout and data processing of linear rates, Anal Biochem. 22, 359-373 (1968). 11. C. SHARMA,R. MANJ~SHWARand S. WEINHOOSE,Hormonal and dietary regulation of hepatic glucokinase, Advances in Enzyme Regulation 2, 189-200 (1964). 12. G. WEBER, R. L. SINGHAL,N. B. STAMM, M. A. LEA and E. A. FISHER, Synchronous behavior pattern of key glycolytic enzymes: glucokinase, phosphofructokinase, and pyruvate kinase, Advances in Enzyme Regulation 4, 59-82 (1966). 13. V. R. Poa-rER and T. ONO, Enzyme patterns in rat liver and Morris hepatoma 5123 during metabolic transitions, CoM Spring Harbor Symposium on Quantitative Biology 26, 365-372 (1961). 14. H. M. TEPPERMANand J. TEPPERMAN,The hexose-monophosphate shunt and adaptive hyperlipogenesis, Diabetes 7, 468--485 (1958). 15. C. PERAINO, C. LAMARand H. C. PITOT, Studies on the mechanism of carbohydrate repression in rat liver, Advances in Enzyme Regulation 4, 199-217 (1966). 16. M. CIVEN, B. M. TR~tMER and C. B. BROWN, The induction of tyrosine a-ketoglutarate and phenylalanine pyruvate transaminases in liver by glucagon, Federation Proc. 26, 347 (1967). 17. E.A. KHAmALLAHand H. C. PITOT, 3'5'-Cyclic AMP and the release ofpolysome-bound proteins in vitro, Biochem. Biophys. Res. Communs. 29, 269-274 (1967).
RNA Synthesis and Serine Dehydratase Induction and Repression More recent studies have demonstrated that doses of high energy T radiation of 400 R or greater significantly inhibit the dietary-induced increase in the activity of this enzyme at periods where such increases are also sensitive to the effects of actinomycin D (4). These data together suggest that initially the induction of the enzyme, serine dehydratase, requires concomitant synthesis of messenger R N A but later synthesis becomes resistant to the effects of actinomycin or 7 radiation and, thereafter, does not require concomitant R N A synthesis. In Fig. 1 is seen an experiment designed to determine the period after initiation of enzyme synthesis by a single dose of * This work was supported in part by grants from the National Cancer Institute (CA 07175) and The American Cancer Society (P-314). I"Special Postdoctoral fellow of the North Atlantic Treaty Organization. ~:Postdoctoral fellow of the National Cancer Institute (CA 21666). § Career Development Awardee of the National Cancer Institute (CA-29,405). 171
172
HANS D. S~LING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
casein hydrolysate during which further enzyme synthesis is resistant to the effects of the antibiotic. It should be noticed that enzyme induction remained completely sensitive to the effects of actinomycin for the first hour and onehalf after the initial intubation. Until the eighth to ninth hour after enzyme SDH UNITS 7oc
60C
/.
50C
~oo
I
,..°,...,
/
/ I
300
200
IOC
\
/!
j/"
,
,...." / //L..'" / / / / " ~
""
l
....... D.D. + ACT D -D.D. ONLY
- - - BASAL SINGLE DOSE CURVE
TIME, HOURS
FIo. 1 Actinomycin D (ACT D) sensitivity and resistance of serine dehydratase induction after an initial single dose of I g of caseinhydrolysate. The format of the experiment is described in ref. 5. At each time point noted by white circles, groups of animals were givena second dose (D.D.) of caseinhydrolysate with and without actinomycin.
induction was initiated, further induction of the enzyme is completely resistant to the effects of the antibiotic. After the ninth hour, however, further enzyme induction is again sensitive to actinomycin administration. Similar, if not identical, data have been obtained in our laboratory by the use of 3/ radiation (unpublished observations). During the period of actinomycin D resistance no R N A synthesis is required indicating that genetic translation is the only process occurring during this time period. For the synthesis of any enzyme the period of actinomycin D resistance has been termed "the template lifetime", indicating that during this period one is seeing the expression of a stable messenger R N A template. As indicated in the introduction, studies (2) demonstrated that the early administration of glucose completely prevented the casein hydrolysate-
HORMONES AND GLUCOSE REPRESSION
173
induced levels of serine dehydratase. I n addition, administration of glucose during periods when enzyme synthesis proved resistant to actinomycin also prevented any further increase in the level of the enzyme. Recent studies using quantitative i m m u n o c h e m i c a l techniques (6) have verified these conclusions showing that in fact glucose shuts off the synthesis of serine dehydratase both at periods of actinomycin D resistance as well as actinomycin D sensitivity (Table 1). It can be noted from the data in Table 1 that if TABLE 1 INDUCTIONAND REPRESSIONOF SERINEDEHYDRATASEIN LIVERSOF INTACTRATS* l*C-Valine incorporation No. of animals
Protein control, 0 Amino acids 6 hr 10 hr 18 hr Amino acids + glucose (10 hr) Amino acids (10 hr) + delayed glucose (6 hr only) L-Tryptophan (10 hr) Glucagon (10 hr)
Serine dehydratase activity
Serine dehydratase/g liver
Soluble proteins/g liver
units/g liver 4.4 -4- 0.4
d.p.m. 66.0 -4- 23.0
320,000
60.0 -4- 14.3 125.6 + 27.1 260.0 4- 20.8
836.0 4- 137.0 1816.0 4- 396.0 1850.0 + 427.0
325,000
5.6 -4- 0.5
40.5 4- 24.3
324,000
43.6 4- 13.4 71.9 4- 6.1 509.7 + 57.0
150.1 4- 45.9 1365.0 -4- 60.0 5858.0 4- 706.0
489,000 273,000 465,000
* The experimental details may be found in ref. 6. An incomplete mixture of essential amino acids, lacking valine and methionine, was administered at the times indicated. L-Tryptophan, 0.37 mmole, was administered at 0 time and 6 hr to the group receiving this single amino acid. Glucagon was administered intraperit0neally at a dose of 0.2 mg/100 g body weight at 0 time and again at 6 hr. The hours in parentheses denote the time of death in relation to 0 time, with the exception of the "6 hr only" designation for delayed glucose which signifies the only time of glucose administration. glucose is given at zero time and continued throughout the experimental conditions an abrupt and complete cessation of the enzyme synthesis occurs. If the administration of glucose is delayed until 6 hr after the administration of the initial dose of a m i n o acids there is a m a r k e d inhibition of the rate of enzyme synthesis as measured at 10 hr. If this is c o m p a r e d with the rate of enzyme synthesis either at 6 to 10 hr and corrected for the total incorporation of amino acid into soluble protein, glucose administration has inhibited enzyme synthesis by 9 5 % . Therefore, these experiments demonstrate quite conclusively that a repression of serine dehydratase synthesis
174
HANSD. SOLING,JOELKAPLAN,MARIEERBSTOESZERAND HENRYC. PITOT
mediated by glucose occurs at the level of genetic translation. Furthermore, in Table 1 one may see that there is an actual increase in the rate of enzyme synthesis between the 6- and 10-hr period. At both of these points under the conditions of this experiment enzyme synthesis would be resistant to actinomycin D. It may also be noted from the data of Table 1 that the hormone, glucagon, at a level of 0.2 mg per 100 g of body weight induces a dramatic increase in both activity and radioactivity of serine dehydratase within 10 hr. Previous studies by Peraino and Pitot (2) have demonstrated that glucagon given in doses used in this experiment was as efficient in the induction of serine dehydratase as was the administration of amino acid mixtures. Furthermore, as with induction by amino acids, shortly after the initiation of glucagon administration enzyme synthesis became resistant to the effects of actinomycin D in much the same manner as after amino acid administration. As seen from the data of Table 1 glucagon induces the greatest rate of synthesis of those materials utilized. Although the data are not shown in this Table, glucose will repress glucagon-induced synthesis of serine dehydratase, although not as efficiently as when amino acids are the inducing agents.
Serine Dehydratase Induction in Adrenalectomized Animals Previous studies (1) have shown that the feeding of a 90% casein diet or the administration of casein hydrolysate resulted in significant increases in
TABLE2 INDUCTION AND REPRESSION OF SERINE DEHYDRATASE AND HORMONES IN LIVERS OF ADRENALECTOMIZED RATS*
14C-Valine incorporation No. of animals
Protein (0 %) Amino acid (6 hr) Amino acid + glucose (6 hr) Glucagon (6 hr) Hydrocortisone (6 hr) Fasting (6 hr) Fasting (18 hr)
Enzyme activity
Serine dehydratase/g liver
Soluble proteins/g liver
units/g liver 40.0 4- 15.0 301.0 4- 18.0
196 4- 52 1481 4- 96
d.p.m. 179,000 4- 18,000 241,000 4- 17,000
161.0 4- 9.0 583.0 -4- 24.0
6604-117 27354-205
239,000 ! 19,000 253,000 + 20,000
323.0 + 11.0 139.0 4- 16.0 250.5 -4- 45.9
1944:t:238 9704-151 14424-199
267,000 ± 19,000 197,000 :k 18,000 123,000 4- 15,000
* See legend of Table 1 for details. Hydrocortisone (10 mg) was injected intraperitoneally at 0 time.
HORMONES AND GLUCOSE REPRESSION
175
serine dehydratase activity in the livers of adrenalectomized rats. In addition, cortisone administration effected a marked rise in serine dehydratase levels of rats maintained on a laboratory chow diet over the period of one week. Studies by Rosen and Nichol (7) have also demonstrated that the acute administration of cortisone will lead to dramatic increases in serine dehydratase activity within 24 hr. Fasting of adrenalectomized animals has also been shown to result in a marked rise in serine dehydratase activity in liver (8). As seen from the data of Table 2 amino acid administration induces a significant rise in the rate of synthesis of serine dehydratase in liver. This rise is markedly inhibited by glucose although not as efficiently as seen in the intact a n i m a l Again, the administration of glucagon appears to be the most efficient inducer of serine dehydratase in the liver of the adrenalectomized rat. Hydrocortisone, in confirmation of other studies (7), is seen to induce a significant rise in the rate of synthesis of this enzyme, almost as great as that seen after amino acid administration. Fasting, as has been reported, also increases both enzyme activity and the rate of synthesis.
The Role of Insulin in Glucose Repression in Rat Liver As was reported earlier (2), administration of varying levels of glucose produces an inhibition of serine dehydratase synthesis of varying degrees. Even at a level of 0.5 g of glucose per dose there is a dramatic inhibition of serine dehydratase synthesis. In more recent studies, seen in Fig. 2, it may 3000 e-,.e CH, o - - o C.H. +GLUC. (20rag) CH. ,I- GLUC. (50rag)
25oo
.L
~
c.,. + GLUC.(Jso~Q)
H
C.H. + GLUC.(3OOmg)
J I
/
~/o
~ 2000 1500
1000
50O
0
I 6
I 12
I 17
FIG. 2 The effect of low doses of glucose on the induction of serine dehydratase by casein hydrolysate. The format of the experiment is described in ref. 2, the only difference being the dosages of glucose utilized as may be seen in the figure.
176
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
be noticed that even lower levels of glucose effectively prevent the increase in serine dehydratase reported earlier. As little as 150 nag of glucose given at 6-hr point effectively causes a reduced rate of increase in serine dehydratase activity. At quite low levels of glucose (50 mg), as seen from the graph, there appears to be an initial stimulation of the rate of serine dehydratase increase. Although the differences noted here are not statistically significant, these data are in line with those seen in yeast wherein glucose at low levels (10-4M) actually stimulates the synthesis rather than inhibits that of the inducible enzyme, ct-glucosidase (J.-P., Jost, unpublished results). The actual mechanism for this stimulation by glucose is not immediately apparent although the previous finding reported from this laboratory that administration of glucose actually increases A T P levels in liver suggests that such a mechanism may account for these data. Since the administration of glucose to animals stimulates a dramatic release of insulin from the pancreas, it is reasonable to suggest that this hormone plays a major role in the effect of glucose seen in these experiments. This is especially true since Suda and his associates (9) have demonstrated that serine dehydratase levels in liver of alloxan-diabetic rats are markedly higher than that seen in normal animals. Administration of low protein, high carbohydrate diets did not appear to alter the level of this enzyme in the liver of alloxan-diabetic animals. In order to substantiate and extend Suda's studies, rats were made diabetic by the intravenous administration of alloxan (45 m g / k g male rat) and held for one week. At the end of this time blood sugars were determined and only those animals exhibiting a fasting blood sugar of greater than 300 mg% were used for the experiments reported here. All animals were placed in rooms lighted from 9 p.m. to 9 a.m. and maintained in the dark for the daylight periods. Food was given to the animals at 9 in the morning and removed at 5 in the afternoon. For the forced-feeding studies animals were intubated with casein and other materials at 8-hr intervals beginning at 12:00 noon after an overnight fast. Animals were sacrificed 24 hr after the initial intubation. Insulin was administered at a dose of 2 units every 2 hr the day before sacrifice and once on the day of sacrifice 6 units of Lente insulin were also given the night before. All enzyme analyses were carried out with an automated system that had been previously described (10). Enzyme units are defined as /xmoles of product formed per hour. As seen from the data of Table 3 which includes measurement of the enzymes serine dehydratase and ornithine transaminase induced after maintenance of animals on a 12% protein diet, administration of casein hydrolysate over a 24-hr period results in a marked gtimulation in the level of both enzymes. Furthermore, the administration of glucose markedly inhibits this increase in the case of serine dehydratase and to a lesser extent in the case of ornithine transaminase. However, the administration of casein with and
HORMONES AND GLUCOSE REPRESSION
177
TABLE3 SERINE DEHYDRATASE (SDH) AND ORNrrmNE TRANSAMINASE (OT) LEVELS IN NORMAL AND DIABETIC LIVER*
SDH
Control (12 Yoprotein diet) Control -t- casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin q- glucose
OT
Normal
Diabetic
Normal
Diabetic
4.9 d- 0.8 33.2 4. 4.0 6.3 4. 0.8 5.7 4- 1.0
24.0 q- 3.1 16.6 ± 1.8 20.5 4. 2.0 30.0 4- 4.2 26.5 4- 2.2
1.5 -4- 0.2 7.7 4. 1.5 6.7 4. 0"8 6.6 4. 0.6
6.1 4. 0.6 8.2 4. 0.7 6.2 + 1.0 6.8 4. 0.4 5.7 4. 0.8
16.7 4- 2.4
3.8 q- 0.4
* All animals (8-12 rats per condition) were maintained on a 12~ protein diet as described in the text. Casein hydrolysate (casein) was administered via stomach tube (I g/4 CO with and without glucose or fructose (2 g/dose) every 8 hr as described in the text. See text for insulin dosages. Enzyme activities are expressed as #M product formed/hr/liver x 103 4standard error of the mean. without glucose and insulin or fructose has little or no effect on the level of these enzymes in the diabetic liver. It should be noted that in support of Suda's data the level of serine dehydratase in the liver of an animal maintained on a 12% protein diet is quite high, being almost at the level induced by casein alone. A possible exception to this is seen in the case of the animal given casein, insulin and glucose wherein ornithine transaminase appears to be repressed. Thus, it would appear that the alloxan-diabetic animal maintained under these conditions exhibits very little, if any, effect of insulin a n d / o r glucose and fructose. Since the administration was over a 24-hr period and previous studies have demonstrated that the half-life of serine dehydratase is about 5 hr (6), it would seem that if repression did occur it should easily be discernible under the conditions of these experiments. A question remains as to whether or not these animals are, in fact, diabetic and this was verified on the basis of blood sugar (greater than 300 nag %) as well as the data seen in Table 4. In Table 4 the levels of glucokinase in these animals are demonstrated. As can be seen in the alloxandiabetic animals, the level of glucokinase is quite low. However, when insulin or insulin and glucose is given, there is a dramatic increase in the level of this enzyme. Similar results may be noted from the data of Table 5 wherein the activities of the citrate cleavage enzyme and glucose 6-phosphate dehydrogenase are seen. In this instance, in normal animals the administration of casein plus glucose or fructose results in a significant increase in the level of both of these enzymes. Such an increase is borderline, if there is any at all in the case of the alloxan-diabetic liver. However, when
178
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT TABLE 4 GLUCOKINASE LEVELS IN NORMAL AND DIABETIC LIVER*
Normal 27.9 23.2 34.1 45.7
Control (12 ~ protein diet) Control + casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin + glucose
4- 3.2 4- 1.3 4- 1.2 4- 4.0
Diabetic 3.3 4.7 6.1 7.8 27.8 57.5
4- 1.0 ± 1.8 4- 2.4 4- 1.7 ± 1.9 4- 3.9
* See legend of Table 3 for details. Enzyme activities are expressed as/~M product formed/hr/liver × 102 4- standard error of the mean.
TABLE 5 CITRATE CLEAVAGE ENZYME (CCE) AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE
(G6PD) LEVELSIN NORMALAND DIABETICLIVER* G6PD
CCE
Control (12~ protein diet) Control + casein Control + casein + glucose Control + casein + fructose Control + casein + insulin Control + casein + insulin + glucose
Normal
Diabetic
18.8 4- 3.1 25.0 q- 2.5 49.8 4- 4.1 42.1 + 3.4
9.8 4- 1.6 8.3 4- 1.0 10.6 4- 1.7 12.4 4- 1.9 44.4 q- 8.4 60.0 4- 6.0
Normal 22.8 46.2 96.8 90.2
± 4.4 4- 5.9 4- 7.4 4- 6.0
Diabetic 9.7 4- 0.8 10.0 4- 1.0 17.9 ± 3.5 20.2 ± 3.3 86.8 4- 15.8 121.54-16.9
* See legend of Table 3 for details. Enzyme activities are expressed as/~Mproduct formed/ hr/liver x 102 4- standard error of the mean. i n s u l i n is given a d r a m a t i c increase occurs in the level of these enzymes in diabetic liver. I n T a b l e 6 is n o t e d the effect of feeding to alloxan-diabetic rats a 90% protein diet together with glucose in the d r i n k i n g water as well as the a d m i n istration of i n s u l i n with a n d without glucose. Perhaps the most striking thing seen with these data is the extremely high level of both serine dehydratase a n d o r n i t h i n e t r a n s a m i n a s e . This m a y be the result of a n extreme s t i m u l a t i o n of gluconeogenesis which is n o r m a l l y present in the diabetic a n d a u g m e n t e d b y the feeding of a high-protein diet. T h e a d m i n i s t r a t i o n of insulin for a 2-day period to these a n i m a l s produces n o significant change in the level of the enzyme, although again, based on half-life studies, if this h o r m o n e h a d been effective changes should be d e m o n s t r a b l e over the time period studied. However, the a d m i n i s t r a t i o n of glucose alone or with i n s u l i n
179
HORMONES AND GLUCOSE REPRESSION TABLE 6 ENZYME LEVELS IN LIVERS OF DIABETIC RATS FED A 90~o PROTEIN DIET* SDH Control 0ontrol + insulin Control + glucose (ad lib) Control + glucose (ad lib) 4- insulin
OT
GK
249.0 4- 21.5 105.7 :k 12.0 2.3 4- 0.5 236.8 4- 26.4 99.3 4- 8.2 30.8 4- 5.7 69.5 4- 26.7
48.0 :k 10.0
6.8 4- 3.5
99.7 4- 14.7
38.1 4- 10.1 37.9 4- 2.9
CCE
G6PD
9.2 4- 0.7 I0.1 4- 1.6 21.9 4- 4.4 24.7 4- 6.0 15.1 4- 1.9
13.1 4- 2.4
37.4 4- 5.4 49.2 4- 11.6
* See text for experimental details. All enzyme activities are expressed as/~M product/hr/ liver × 102 4- standard error of the mean except SDH wherein the factor is 103.
simultaneously with the 90% protein diet does result in significant inhibition of the high levels of both of these enzymes. These results suggest that glucose alone is of much greater significance in the mechanism of glucose repression than is the hormone insulin--at least with respect to these two enzymes. In the case of glucokinase, citrate cleavage enzyme and glucose6-phosphate dehydrogenase, as expected from earlier results, only animals given insulin showed a highly significant increase in the level of these enzymes. As an adjunct to these studies, the effect of diabetes and glucose or fructose with and without insulin on the levels of tryptophan pyrrolase and tyrosine transaminase was ascertained. As seen from the data of Table 7 the TABLE 7 TRYPTOPHAN PYRROLASE (TP) AND TYROSINE a-KETOGLUTARATE TRANSAMINASE (TAKG) LEVELS IN NORMAL AND DIABETIC LIVER*
TP (units/liver) 12 ~ Protein diet Control Control + casein Control + casein Control + casein Control + casein Control + casein
+ + + + +
glucose fructose insulin insulin glucose
90 ~o Protein diet Control Control + insulin Control + glucose (ad lib.) Control + glucose (ad lib.) + insulin
T A K G (units/liver No 2)
Normal Diabetic 22.8 4- 8.4 85.1 4- 6.0 86.3 4- 11.5 121.8 4- 13.1 87.4 + 8.7 148.6 4- 19.5 104.9 4- 24.2 76.4 4- 10.9 125.4 4- 17.6 84.5 4- 9.8 152.4 220.7 120.4 86.9
4444-
15.0 21.6 34.9 13.4
*See legends to Tables 3 and 6 for experimental details. G
Normal 5.5 4- 0.8 17.4 4- 5.9 25.6 4- 5.7 22.0 4- 2.6
Diabetic 9.2 ± 2.4 19.6 4- 3.7 22.3 4- 7.1 11.2 4- 1.2 22.3 4- 4.9 16.4 4- 1.9 53.5 47.6 37.9 18.7
4444-
2.4 1.9 7.5 5.3
180
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
effects of these dietary and hormonal conditions are not readily related to either variable. Administration of casein hydrolysate, as has been earlier demonstrated, results in a dramatic stimulation of the activity of both enzymes. However, glucose or fructose administration has essentially no effect on this in substantiation of earlier results with tyrosine transaminase wherein administration of casein plus carbohydrate actually stimulated the level of this enzyme under certain conditions. In the diabetic liver casein itself appears to cause a significant increase in the level of tyrosine transaminase which can be eliminated by the administration of fructose. Insulin and glucose, however, appear to have little or no effect. Similar results are seen with tryptophan pyrrolase, wherein also insulin plus glucose produces a significant inhibition of the slight increase produced by casein. When feeding a 90% protein diet, only the administration of glucose and insulin produces an inhibition. DISCUSSION
As can be seen from the data of the last five tables, the induction of a number of enzymes is considerably altered by the production of experimental alloxan diabetes. In general, the effects seen may be grouped by enzymes into several groups. Serine dehydratase and ornithine transaminase exhibit little effect of glucose casein or insulin in the liver of the alloxan diabetic animal. Administration of a high-protein diet, however, results in dramatic increases which are prevented by simultaneous glucose administration in the drinking water, but not affected by a 48-hr administration of insulin. This would strongly suggest that the effect of glucose in repressing the synthesis of serine dehydratase is not primarily mediated by insulin, but is probably an extremely complex mechanism which involves several hormones such as glucagon, hydrocortisone, and possibly others. Experiments are now in progress to determine the effect of hypophysectomy in association with alloxan diabetes. If the diabetic animal is adrenalectomized, the levels of serine dehydratase are essentially the same as those seen in the intact diabetic rat. The responses of glucokinase seen in this experiment completely parallel those reported in the literature by a number of other authors (11,12). In the case of the citrate cleavage enzyme and glucose 6-phosphate dehydrogenase the effects are not quite so simple as those seen with glucokinase. Specifically the administration of carbohydrate and protein does result in a significant rise in the level of both of these enzymes. Earlier studies by Ono and Potter (13) had demonstrated that adrenalectomy prevented the fasting-refeeding induction of glucose 6-phosphate dehydrogenase that had been reported by Tepperman (14). In addition, in this latter enzyme there does appear to be an effect of carbohydrate and protein even in the liver of the diabetic animal. A similar, although less significant, increase may be seen in animals fed a 90% protein diet with glucose in their drinking water. Finally, the effects of
HORMONES AND GLUCOSE REPRESSION
181
these various agents on tryptophan pyrrolase and tyrosine transaminase do not appear to fit into any specific pattern. Previous studies had already demonstrated the "anomalous" response of tyrosine transaminase to glucose and adrenal steroids (15). Recent studies by Civen and his associates (16) suggesting that cyclic 3"5"-AMP is important in the regulation of the level of this enzyme are quite significant, particularly on the basis of studies from this laboratory (17) as well as several others. It is possible that this small molecule plays a major role in the changes in enzyme levels that we have described here, but as yet what this role is remains to be elucidated. SUMMARY
Previous studies from this laboratory have demonstrated that the administration of amino acid mixtures to rats previously fed a 0% protein diet will markedly induce the synthesis of several hepatic enzymes. In particular, serine dehydratase is markedly induced under these conditions. The induction is characterized by an initial period of RNA synthesis, but thereafter appears to continue independent of RNA synthesis. That the increases seen are the result of actual changes in the rate of synthesis of the enzyme has been demonstrated by immunochemical methods. Relatively low doses of glucose (150 mg) effectively repress enzyme synthesis under these conditions. Glucagon also induces serine dehydratase, this induction being repressed by glucose. Insulin given to animals partially repressed with glucose has no further effect. However, rats made diabetic with alloxan show marked increases in the level of serine dehydratase, this high level being unaffected by glucose, fructose, or insulin administration. Similar findings may be seen with the mitochondrial enzyme, ornithine transaminase. The enzymes of carbohydrate metabolism, glucokinase, citrate cleavage enzyme and glucose 6-phosphate dehydrogenase, may be induced by protein and glucose in the intact animal, but appear to require glucose and insulin in the diabetic animal. Animals fed a high protein diet show marked increases in serine dehydratase and ornithine transaminase which is not affected by the chronic administration of insulin. These studies demonstrate that glucose repression is a complex phenomenon, probably requiring the interaction of hormones and substrates. The data seen in this paper, however, do not in any way support a primary role for insulin in the mechanism of glucose repression in rat liver. REFERENCES 1. H. C. PITOT and C. PERAINO,Studies on the induction and repression of enzymes in rat liver I. Induction of threonine dehydrase and ornithine-8-transarninase by oral incubation of casein hydrolysate, J. Biol. Chem. 239, 1783-1788 (1964). 2. C. I~RAINO and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver II. Carbohydrate repression of dietary and hormonal induction of threonine dehydrase and ornithine-8-transaminase, J. BioL Chem. 239, 4308-4313 (1964).
182
HANS D. SOLING, JOEL KAPLAN, MARIE ERBSTOESZER AND HENRY C. PITOT
3. H. C. PITOT and C. PE~dNO, Carbohydrate repression of enzyme induction in rat liver, J. Biol. Chem. 238, PC1910-12 (1963). 4. H. C. PiTOT, C. PERAINO.C. LAMARand S. L~HER, The effect of gamma radiation on the dietary and hormonal induction of enzymes in rat liver, Science 150, 901 (1965). 5. H. C. PITOT, C. PERAtNO, C. LAMARand A. L. I~NNAN, Template stability of some enzymes in rat liver and hepatoma, Proc. NatL Acad. Sci. 54, 845-851 (1965). 6. J. P. JOST, E. A. KHAmALLAHand H. C. PITOT, Studies on the induction and repression of enzymes in rat liver V. Regulation of the rate of synthesis and degradation of serine dehydratase by dietary amino acids and glucose, J. Biol. Chem. 243, 3057-3066 (1968). 7. F. ROSEN and C. A. NICHOL, Studies on the nature and specificity of the induction of several adaptive enzymes responsive to cortisol, Advances in Enzyme Regulation 2, 115-135 (1964). 8. H. C. PITOT, Studies on the control of protein synthesis in normal and neoplastic rat liver, P h . D . Dissertation, Tulane University, (1959). 9. E. ISHIKAWA,P. }.~IINIGAWAand M. SODA, Hormonal and dietary control of serine dehydratase in rat liver, J. Biochem. (Japan) 57, 506-513 (1965). 10. H. C. PITOT, N. WRAr'rEN and M. POmIER, The automated assay of complete enzyme reaction rates II. Digital readout and data processing of linear rates, Anal Biochem. 22, 359-373 (1968). 11. C. SHARMA,R. MANJ~SHWARand S. WEINHOOSE,Hormonal and dietary regulation of hepatic glucokinase, Advances in Enzyme Regulation 2, 189-200 (1964). 12. G. WEBER, R. L. SINGHAL,N. B. STAMM, M. A. LEA and E. A. FISHER, Synchronous behavior pattern of key glycolytic enzymes: glucokinase, phosphofructokinase, and pyruvate kinase, Advances in Enzyme Regulation 4, 59-82 (1966). 13. V. R. Poa-rER and T. ONO, Enzyme patterns in rat liver and Morris hepatoma 5123 during metabolic transitions, CoM Spring Harbor Symposium on Quantitative Biology 26, 365-372 (1961). 14. H. M. TEPPERMANand J. TEPPERMAN,The hexose-monophosphate shunt and adaptive hyperlipogenesis, Diabetes 7, 468--485 (1958). 15. C. PERAINO, C. LAMARand H. C. PITOT, Studies on the mechanism of carbohydrate repression in rat liver, Advances in Enzyme Regulation 4, 199-217 (1966). 16. M. CIVEN, B. M. TR~tMER and C. B. BROWN, The induction of tyrosine a-ketoglutarate and phenylalanine pyruvate transaminases in liver by glucagon, Federation Proc. 26, 347 (1967). 17. E.A. KHAmALLAHand H. C. PITOT, 3'5'-Cyclic AMP and the release ofpolysome-bound proteins in vitro, Biochem. Biophys. Res. Communs. 29, 269-274 (1967).