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Hepatic gluconeogenesis in rats treated with cortisol
GENER4L
.4ND
CGMP.4R.4TIVE
ENDOCRINOLOGY
Hepatic
10,
434-437
Gluconeogenesis
D. BELLAMY, Department
in Rats Treated
RUTH
of Zoology.
(1968)
A. LEONARD, The
Received
I.uitlersity,
An’D
Sheffield,
December
with KAY
Cortisol DVLIEU
S. 10, ,?TK
Et~gln~~tl
20, 1967
A single subcutaneous injection of co&o1 into starved rats increased liver glycogen after a short lag period, and radioactivity derived from glucose-“C injected at the same time as cortisol appeared in blood glucose and glycogen. At the time corresponding to the onset of glycogen deposition there was an increased rate of incorporat,ion of ‘“C into glycogen but a fall in the specific activity of the glycogen tha.t was not paralleled by a drop in the specific activity of blood glucose. In contrast to these results, there was very little incorporation of radioartivity derived from injected lactate or glycerol. Using labelled alanine, it was concluded that there m-as no large influx of amino acids into the plasma from peripheral tissues. In order to explain the fall in the specific activity of glycogen it is suggested that one of the initial effects of cortisol on liver results in an increased rate of gluconeogenesis from liver protein.
The observation of Long, Katzin, and Fry (1940) t,hat corticosteroids of the cortisol t.ype increased the amount of liver glycogen has been confirmed many times. It is clear that. amino acids, arising from the involution of peripheral tissues, and bIood glucose both contribute to the newly formed glycogen (Ashmore, 1960; Long, Fry, and Bonnycastle, 1960). The present work was undert’aken in order to assess the relative importance of plasma glucose and amino acids in glycogen synthesis shortly after treatment of starved rats with cortisol. MATERIALS
AND
clamping.” The body cavity was quickly opened and a lobe of liver in situ was pressed between stainless steel tongs cooled to -lo”, and lifted from the animal. Glycogen. Liver glycogen was measured by the method of Van der Vies (1954). Radioactive glycogen was extracted by the method of Roe et al. (1961) and precipitated after adding 100 mg rabbit liver glycogen to the trichloroacetic acid extract. The precipitate was washed three times on t,he centrifuge with ethanol and dissolved in 1.5 ml water. Radioactivity was measured in a scintillation counter (1.0 ml glycogen solution plas 10 ml dioxan POPOP (cf. Bellamy et al., 1962). Quenching varied from 85 to 94% and counts were corrected to 90-‘/o quenching. G&ose. Glucose was added to plasma to bring the concentration to 2% w/v and samples werr chromatographed as described by Bacon and Bacon (1954). Glucose was eluted and measured as described for glycogen. Endogenous plasma glucose was measured directly by the method of Nelson (1944). Alanine. Plasma alanine was determined after paper chromatography of 5% trichloroacetic acid extracts (Bellamy, 1961). Alanine was eluted and measured as for glycogen.
METHODS
Animals. Male albino rats, l-month-old, were taken fro.m a randomly mated colony in the Department of Zoology, University of Sheffield. In all experiments, they were deprived of food from 17.06 hours to 09.30 hours and injected subcutaneously with cortisol suspension (0.5 mg/ 0.5 ml 0.9% NaCl containing 1% carboxymethylcellulose), containing 5 pc uniformly labelled glucose-Y, lactate, or alanine. When glycerol was used it was labelled at C-l. Blood was obtained by decapitation without stunning, and collected through a heparinieed funnel (rinsed with heparin solution, 500 U/ml, and dried at 2CW). Liver samples were taken as nearly as possible to the time of decapitation by the technique of “freeze
RESULTS
When rats were deprived of food overnight, liver glycogen, normally equivalent to about 8% of the wet weight of liver, fell 434
CORTISOL
INCORPORATION
Time (minutes)
435
GLUCONEOGENESIS
TABLE OF GLUCOSE-W
1 INTO
LIVER
GLYCOGEN~
Glyoogenb (per 100 gm) 10-B (counts/minute)
-
10 20 30 60 90 120
AND
5Y3 636 730 844 1375 2070
a Rats were injected with analysis carried out on liver b + SE for six animals. c For four animals.
+ k It f + +
21 22 20 24 106 114
Specific activity 10-a (counts/minute/Jmole (mmoles
0.51 0.46 0.50 0.75 1.55 2.90 4.11
glucose)
f 2~ f AI + f +
0.03 0.02 0.02 0.01 0.04 0.06 0.16
0.5 mg cortisol and 5 PC glucose-U-W and plasma as described in the text.
to below 1%. A single subcutaneous injection of cortisol suspension increased the concentration of glycogen after a lag period of about 30 minutes (Table 1). Two hours after injection, the concentration of glycogen was about 0.7% of wet weight. When glucose-‘% was injected subcutaneously together with cortisol, glucoseJ4C was found in plasma 20 minutes after the time of injection. Thereafter, the concentration of radioactive glucose dropped steadily, and after 2 hours had reached 10% of the initial value. There was a rapid appearance of radioactivity in glycogen within 10 minutes of injection (specific activity 16% of glucose). The amount of 14C increased gradually for another 20 minutes. The possibility that this radioactivity was due to the adsorption of glucose or one of its metabolites was checked by repeatedly dissolving the glycogen in solutions containing glucose and amino acids and then reprecipitating the carbohydrate. This treatment did not alter the content of 14C. At the time corresponding to the onset of glycogen deposition there was an increased rate of incorporation of radioactivity into glycogen. Radioact,ivity continued to accumulate over the 2-hour experimental period, but from 30 to 90 minutes the specific activity of t,he glycogen fell by about 50%. Between 90 and 120 minutes the specific activity increased slightly and at the end of the experiment it was about 70% of that of blood glucose. There was no
Glycogen
glucose) GlUC0-W
-
-
1.27 1.27 0.97 0.54 0.47 0.50
7.94 7.66 6.20 5.05 3.11 0.71
(150 Fc/mmole),
killed
at intervals,
and
change in the concentration of plasma glucose over the whole period. Very little radioactivity was found in glycogen after the injection of labelled lactate or glycerol. The largest quantities of ‘“C were observed 30 minutes after injection; there was then a drop in radioactivity for the rest of the experimental period (Table 2). TABLE 2 ITXCORPORATION OF RADIOACTIVITY DERIVED FROM LACTATE-W AND GLYCEROL INTO LIVER GLYCOGEN” Liver glycogen
Time (minutes)
10 20 30 60 90 120
10-z (counts/minute/lOOh Lactate
Not
detectable 22 + 6 42 f S 40 ?c 10 31 k 9 22 * 4
a The experiment was carried Table 1. Animals were injected lactate (U-W, 100 &mmole) 150 &mmole). * SE for six animals.
gm liver) Glycerol
Not
detectable 57 k 5 77 + 4 73 -t 5 54 i- 6 43 lk 5
out as described in with 5 PC sodium or glycerol (1-W;
The concentration of radioactive alanine in plasma derived from alanine-14C injected subcutaneously, dropped by about 85% in the 2 hours following injection. Treatment with cortisol (injected at the same time as the alanine) did not affect the rate of
436
BELLAMY,
EFFECT
LEONARD,
OF CORTISOL
AND
TABLE 3 ON ALANINE-%
DULIEU
IN PLASMA’”
Alanine concentrations 101 (counts/minute/ml) Tiie (minutes)
15 17 30 32 60 62 118 120
Normal
7.52
2.20
+ 0.47 zk 0.30 + 0.25
0.90
* 0.33
3.73
Alanine specific activityb 10-a (counts/minute/rmole) Cortisol
7.19 2.67
NOrlId
74.0
* 3.2
3’2.4
* 1.9
+ 0.76 f 0.35
Cortisol
71.8
* 4.1
22.6
3~ 1.9
20.2 13.0
k 1.7 + 1.3
20 7 * 1.8 1.97 1.25
f 0.13 + 0.25 -
10.5
rt 1.1
0 Rats were injected with 5 ~c uniformly labelled Galanine (10 mc/mmole) wit,h and without 0.5 mg cortisol. Blood was collected at intervals and the alanine-14C concentration determined as in the text,. * + SE for six animals. disappearance of radioactive alanine from plasma (Table 3). There was no change in the concentration of plasma alanine during the experimental period. DISCUSSION
A previous study showed that 4 hours after treatment with cortisol there was no difference in the specific activity of liver glycogen formed from radioact,ive glucose when compared with that in rats not treated with esteroid glucose (Ashmore, 1960). Thus, gluconeogenesis was ruled out as an important source of glycogen at this time. The present net results are in agreement with these conclusions in that 2 hours after injection, t,he specific activity of liver glycogen was approaching t,hat of blood glucose. However, 90 minutes earlier, when liver glycogen first began to increase, there was a drop in the specific activity of glycogen indicative of a relatively large influx of compounds not in equilibrium with blood glucose. Leaving aside blood glucose, plasma does not contain sufficient glycogenic materials to account for the extra glycogen deposited. Taking into account the known actions of cortisol in zlivo, there are three possible extrahepatic sources of unlabelled compounds that may have been responsible for the fall in the specific activity of glycogen: (1) glycerol released from adipose tissue, (2) lactic acid from muscle metab-
olism and (3) amino acids derived from cell protein. The generalization may be made that hormones of the cort.isol type prevent the mobilization of fats in the starved rat (Stoerk and Porter, 1950; Levy and Ramey, 1959). In studies similar to those reported here, it was found that there was very little incorporation of lactic acid into blood glucose, and acute treatment with cortisol had no effect on lactate recycling (Ashmore, 1960). These conclusions are in accord with the small amounts of radioactivity that were found in glycogen after the injection of labelled lactate or glycerol. As cortisol did not increase t,he turnover of plasma alanine, it is unlikely that glycogen was formed from an extrahepatic source of amino acids. This leaves the possibility that glycogenic amino acids arose in the liver itself. Although liver protein usually increases in amount after cortisol treatment (Goodlad and Munro, 1959; Weber et al.. 1965). this does not always occur in conditions where there is a high concentration of endogenous plasma cort,icosteroids. Thus, it, is known that the process of protein mobilization from the liver of starved rodents involves the adrenal cortex (White, 1947) and an increase in the concentration of plasma corticosterone (Bellamy et al., 1968). Therefore, it may be that the initial effect of injected cortisol on liver is to bring about increased gluco-
CORTISOL
AND
neogenesis at the expense of liver protein, in addition to stimulating glycogenesis from plasma glucose. ACKNOWLEDGMENTS The work was financed largely by a research grant from the Science Research Council (NO. B/SR/2552). The authors wish to thank Professor W. Bartley, who provided facilities for part of the work to be carried out in the Department of Biochemistry, Universit,y of Sheffield. REFERENCES J. (1960). The role of adrenal steroids in the regulation of hepatic metabolism. In “Metabolic effects of adrenal hormones” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 25-37. Churchill, London. BACON, E., AND BACON, J. S. D. (1954). The occurrence of isomaltose among the products of heating glucose in dilute mineral acid. Biochem. .I. 58, 396-402. BELLAMY, D. (1961). The endogenous citric acid cycle intermediates and amino acids of mitochondria. Biochem. J. 82, 2W224. BELL.~MY, D., PHILLIPS, J. G., CHESTER JONES, I., .4ND LEONARD, R. A. (1962). The uptake of cortisol by rat tissues. Biochem. J. 85, 537-545. BELLAMY, D., LEONARD, R. A., DULIEU, K., AND STEVENSON, A. (1968). Starvation metabolism and plasma corticosterone with reference to the actions of metopirone and propylthiouracil. Gen. Comp. Endocrinol. 10, 119-125. GOODLAD, G. A. J., AND MUNRO, H. N. (1959). ASHMORE,
437
GLUCONEOGENESIS
Diet and action of cortisone on protein metabolism. Biochem. J. 73, 343-348. LEVY, A. C., AND RAMEY, E. R. (1959). The effect of adrenal steroids on peripheral fat mobilization in the rat. Endocrinology 64, 586-591. LONG, C. N. H., FRY, E. G., AND BONNYCASTLE, M. (1960). The effect of cortisol on carbohydrate deposition and urea nitrogen excretion in the adrenalectomized rat. Acta Endocrinol. Coppenh.. Suppl. 51, 819. LONG, c. N. H., KATZIN, B., AND FRY, E. G. (1940). The adrenal cortex and carbohydrate metabolism. Endocrinology, 26, 369-334. NELSON, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375-380. ROE, J. H., BAILEY, J. M., GRAY, R. R., AND ROBINSON, J. N. (1961). Complete removal of glycogen from tissues by extraction with cold trichloroacetic acid solution. J. Biol. Chem. 236, 12441246. STOERK, H. C. AND PORTER, C. C. (1950). Prevention of loss of body fat by cortisone. Proc. Sot. Exptl.
Biol.
Med.
74,
65-67.
VAN DER VIES, J. (1954). Two methods for the determination of glycogen in liver. B&hem. J. 57, 410-416. WEBER, G., SRIVASTAVA, S. K., AND SINGHAL, R. L. (1965). The role of enzymes in homeostasis VII. Early effects of corticosteroid hormones on hepatic gluconeogenic enzymes, ribonucleic acid metabolism and amino acid level. J. Biol. Chem.
240,
750-756.
WHITE, A. (1947). Influence of endocrine secretions on the structure and function of lymphoid tissue. Harvey Lectures 1947-48, 43-70.
.4ND
CGMP.4R.4TIVE
ENDOCRINOLOGY
Hepatic
10,
434-437
Gluconeogenesis
D. BELLAMY, Department
in Rats Treated
RUTH
of Zoology.
(1968)
A. LEONARD, The
Received
I.uitlersity,
An’D
Sheffield,
December
with KAY
Cortisol DVLIEU
S. 10, ,?TK
Et~gln~~tl
20, 1967
A single subcutaneous injection of co&o1 into starved rats increased liver glycogen after a short lag period, and radioactivity derived from glucose-“C injected at the same time as cortisol appeared in blood glucose and glycogen. At the time corresponding to the onset of glycogen deposition there was an increased rate of incorporat,ion of ‘“C into glycogen but a fall in the specific activity of the glycogen tha.t was not paralleled by a drop in the specific activity of blood glucose. In contrast to these results, there was very little incorporation of radioartivity derived from injected lactate or glycerol. Using labelled alanine, it was concluded that there m-as no large influx of amino acids into the plasma from peripheral tissues. In order to explain the fall in the specific activity of glycogen it is suggested that one of the initial effects of cortisol on liver results in an increased rate of gluconeogenesis from liver protein.
The observation of Long, Katzin, and Fry (1940) t,hat corticosteroids of the cortisol t.ype increased the amount of liver glycogen has been confirmed many times. It is clear that. amino acids, arising from the involution of peripheral tissues, and bIood glucose both contribute to the newly formed glycogen (Ashmore, 1960; Long, Fry, and Bonnycastle, 1960). The present work was undert’aken in order to assess the relative importance of plasma glucose and amino acids in glycogen synthesis shortly after treatment of starved rats with cortisol. MATERIALS
AND
clamping.” The body cavity was quickly opened and a lobe of liver in situ was pressed between stainless steel tongs cooled to -lo”, and lifted from the animal. Glycogen. Liver glycogen was measured by the method of Van der Vies (1954). Radioactive glycogen was extracted by the method of Roe et al. (1961) and precipitated after adding 100 mg rabbit liver glycogen to the trichloroacetic acid extract. The precipitate was washed three times on t,he centrifuge with ethanol and dissolved in 1.5 ml water. Radioactivity was measured in a scintillation counter (1.0 ml glycogen solution plas 10 ml dioxan POPOP (cf. Bellamy et al., 1962). Quenching varied from 85 to 94% and counts were corrected to 90-‘/o quenching. G&ose. Glucose was added to plasma to bring the concentration to 2% w/v and samples werr chromatographed as described by Bacon and Bacon (1954). Glucose was eluted and measured as described for glycogen. Endogenous plasma glucose was measured directly by the method of Nelson (1944). Alanine. Plasma alanine was determined after paper chromatography of 5% trichloroacetic acid extracts (Bellamy, 1961). Alanine was eluted and measured as for glycogen.
METHODS
Animals. Male albino rats, l-month-old, were taken fro.m a randomly mated colony in the Department of Zoology, University of Sheffield. In all experiments, they were deprived of food from 17.06 hours to 09.30 hours and injected subcutaneously with cortisol suspension (0.5 mg/ 0.5 ml 0.9% NaCl containing 1% carboxymethylcellulose), containing 5 pc uniformly labelled glucose-Y, lactate, or alanine. When glycerol was used it was labelled at C-l. Blood was obtained by decapitation without stunning, and collected through a heparinieed funnel (rinsed with heparin solution, 500 U/ml, and dried at 2CW). Liver samples were taken as nearly as possible to the time of decapitation by the technique of “freeze
RESULTS
When rats were deprived of food overnight, liver glycogen, normally equivalent to about 8% of the wet weight of liver, fell 434
CORTISOL
INCORPORATION
Time (minutes)
435
GLUCONEOGENESIS
TABLE OF GLUCOSE-W
1 INTO
LIVER
GLYCOGEN~
Glyoogenb (per 100 gm) 10-B (counts/minute)
-
10 20 30 60 90 120
AND
5Y3 636 730 844 1375 2070
a Rats were injected with analysis carried out on liver b + SE for six animals. c For four animals.
+ k It f + +
21 22 20 24 106 114
Specific activity 10-a (counts/minute/Jmole (mmoles
0.51 0.46 0.50 0.75 1.55 2.90 4.11
glucose)
f 2~ f AI + f +
0.03 0.02 0.02 0.01 0.04 0.06 0.16
0.5 mg cortisol and 5 PC glucose-U-W and plasma as described in the text.
to below 1%. A single subcutaneous injection of cortisol suspension increased the concentration of glycogen after a lag period of about 30 minutes (Table 1). Two hours after injection, the concentration of glycogen was about 0.7% of wet weight. When glucose-‘% was injected subcutaneously together with cortisol, glucoseJ4C was found in plasma 20 minutes after the time of injection. Thereafter, the concentration of radioactive glucose dropped steadily, and after 2 hours had reached 10% of the initial value. There was a rapid appearance of radioactivity in glycogen within 10 minutes of injection (specific activity 16% of glucose). The amount of 14C increased gradually for another 20 minutes. The possibility that this radioactivity was due to the adsorption of glucose or one of its metabolites was checked by repeatedly dissolving the glycogen in solutions containing glucose and amino acids and then reprecipitating the carbohydrate. This treatment did not alter the content of 14C. At the time corresponding to the onset of glycogen deposition there was an increased rate of incorporation of radioactivity into glycogen. Radioact,ivity continued to accumulate over the 2-hour experimental period, but from 30 to 90 minutes the specific activity of t,he glycogen fell by about 50%. Between 90 and 120 minutes the specific activity increased slightly and at the end of the experiment it was about 70% of that of blood glucose. There was no
Glycogen
glucose) GlUC0-W
-
-
1.27 1.27 0.97 0.54 0.47 0.50
7.94 7.66 6.20 5.05 3.11 0.71
(150 Fc/mmole),
killed
at intervals,
and
change in the concentration of plasma glucose over the whole period. Very little radioactivity was found in glycogen after the injection of labelled lactate or glycerol. The largest quantities of ‘“C were observed 30 minutes after injection; there was then a drop in radioactivity for the rest of the experimental period (Table 2). TABLE 2 ITXCORPORATION OF RADIOACTIVITY DERIVED FROM LACTATE-W AND GLYCEROL INTO LIVER GLYCOGEN” Liver glycogen
Time (minutes)
10 20 30 60 90 120
10-z (counts/minute/lOOh Lactate
Not
detectable 22 + 6 42 f S 40 ?c 10 31 k 9 22 * 4
a The experiment was carried Table 1. Animals were injected lactate (U-W, 100 &mmole) 150 &mmole). * SE for six animals.
gm liver) Glycerol
Not
detectable 57 k 5 77 + 4 73 -t 5 54 i- 6 43 lk 5
out as described in with 5 PC sodium or glycerol (1-W;
The concentration of radioactive alanine in plasma derived from alanine-14C injected subcutaneously, dropped by about 85% in the 2 hours following injection. Treatment with cortisol (injected at the same time as the alanine) did not affect the rate of
436
BELLAMY,
EFFECT
LEONARD,
OF CORTISOL
AND
TABLE 3 ON ALANINE-%
DULIEU
IN PLASMA’”
Alanine concentrations 101 (counts/minute/ml) Tiie (minutes)
15 17 30 32 60 62 118 120
Normal
7.52
2.20
+ 0.47 zk 0.30 + 0.25
0.90
* 0.33
3.73
Alanine specific activityb 10-a (counts/minute/rmole) Cortisol
7.19 2.67
NOrlId
74.0
* 3.2
3’2.4
* 1.9
+ 0.76 f 0.35
Cortisol
71.8
* 4.1
22.6
3~ 1.9
20.2 13.0
k 1.7 + 1.3
20 7 * 1.8 1.97 1.25
f 0.13 + 0.25 -
10.5
rt 1.1
0 Rats were injected with 5 ~c uniformly labelled Galanine (10 mc/mmole) wit,h and without 0.5 mg cortisol. Blood was collected at intervals and the alanine-14C concentration determined as in the text,. * + SE for six animals. disappearance of radioactive alanine from plasma (Table 3). There was no change in the concentration of plasma alanine during the experimental period. DISCUSSION
A previous study showed that 4 hours after treatment with cortisol there was no difference in the specific activity of liver glycogen formed from radioact,ive glucose when compared with that in rats not treated with esteroid glucose (Ashmore, 1960). Thus, gluconeogenesis was ruled out as an important source of glycogen at this time. The present net results are in agreement with these conclusions in that 2 hours after injection, t,he specific activity of liver glycogen was approaching t,hat of blood glucose. However, 90 minutes earlier, when liver glycogen first began to increase, there was a drop in the specific activity of glycogen indicative of a relatively large influx of compounds not in equilibrium with blood glucose. Leaving aside blood glucose, plasma does not contain sufficient glycogenic materials to account for the extra glycogen deposited. Taking into account the known actions of cortisol in zlivo, there are three possible extrahepatic sources of unlabelled compounds that may have been responsible for the fall in the specific activity of glycogen: (1) glycerol released from adipose tissue, (2) lactic acid from muscle metab-
olism and (3) amino acids derived from cell protein. The generalization may be made that hormones of the cort.isol type prevent the mobilization of fats in the starved rat (Stoerk and Porter, 1950; Levy and Ramey, 1959). In studies similar to those reported here, it was found that there was very little incorporation of lactic acid into blood glucose, and acute treatment with cortisol had no effect on lactate recycling (Ashmore, 1960). These conclusions are in accord with the small amounts of radioactivity that were found in glycogen after the injection of labelled lactate or glycerol. As cortisol did not increase t,he turnover of plasma alanine, it is unlikely that glycogen was formed from an extrahepatic source of amino acids. This leaves the possibility that glycogenic amino acids arose in the liver itself. Although liver protein usually increases in amount after cortisol treatment (Goodlad and Munro, 1959; Weber et al.. 1965). this does not always occur in conditions where there is a high concentration of endogenous plasma cort,icosteroids. Thus, it, is known that the process of protein mobilization from the liver of starved rodents involves the adrenal cortex (White, 1947) and an increase in the concentration of plasma corticosterone (Bellamy et al., 1968). Therefore, it may be that the initial effect of injected cortisol on liver is to bring about increased gluco-
CORTISOL
AND
neogenesis at the expense of liver protein, in addition to stimulating glycogenesis from plasma glucose. ACKNOWLEDGMENTS The work was financed largely by a research grant from the Science Research Council (NO. B/SR/2552). The authors wish to thank Professor W. Bartley, who provided facilities for part of the work to be carried out in the Department of Biochemistry, Universit,y of Sheffield. REFERENCES J. (1960). The role of adrenal steroids in the regulation of hepatic metabolism. In “Metabolic effects of adrenal hormones” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 25-37. Churchill, London. BACON, E., AND BACON, J. S. D. (1954). The occurrence of isomaltose among the products of heating glucose in dilute mineral acid. Biochem. .I. 58, 396-402. BELLAMY, D. (1961). The endogenous citric acid cycle intermediates and amino acids of mitochondria. Biochem. J. 82, 2W224. BELL.~MY, D., PHILLIPS, J. G., CHESTER JONES, I., .4ND LEONARD, R. A. (1962). The uptake of cortisol by rat tissues. Biochem. J. 85, 537-545. BELLAMY, D., LEONARD, R. A., DULIEU, K., AND STEVENSON, A. (1968). Starvation metabolism and plasma corticosterone with reference to the actions of metopirone and propylthiouracil. Gen. Comp. Endocrinol. 10, 119-125. GOODLAD, G. A. J., AND MUNRO, H. N. (1959). ASHMORE,
437
GLUCONEOGENESIS
Diet and action of cortisone on protein metabolism. Biochem. J. 73, 343-348. LEVY, A. C., AND RAMEY, E. R. (1959). The effect of adrenal steroids on peripheral fat mobilization in the rat. Endocrinology 64, 586-591. LONG, C. N. H., FRY, E. G., AND BONNYCASTLE, M. (1960). The effect of cortisol on carbohydrate deposition and urea nitrogen excretion in the adrenalectomized rat. Acta Endocrinol. Coppenh.. Suppl. 51, 819. LONG, c. N. H., KATZIN, B., AND FRY, E. G. (1940). The adrenal cortex and carbohydrate metabolism. Endocrinology, 26, 369-334. NELSON, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375-380. ROE, J. H., BAILEY, J. M., GRAY, R. R., AND ROBINSON, J. N. (1961). Complete removal of glycogen from tissues by extraction with cold trichloroacetic acid solution. J. Biol. Chem. 236, 12441246. STOERK, H. C. AND PORTER, C. C. (1950). Prevention of loss of body fat by cortisone. Proc. Sot. Exptl.
Biol.
Med.
74,
65-67.
VAN DER VIES, J. (1954). Two methods for the determination of glycogen in liver. B&hem. J. 57, 410-416. WEBER, G., SRIVASTAVA, S. K., AND SINGHAL, R. L. (1965). The role of enzymes in homeostasis VII. Early effects of corticosteroid hormones on hepatic gluconeogenic enzymes, ribonucleic acid metabolism and amino acid level. J. Biol. Chem.
240,
750-756.
WHITE, A. (1947). Influence of endocrine secretions on the structure and function of lymphoid tissue. Harvey Lectures 1947-48, 43-70.