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Glucagon and ketogenesis
Glucagon and Ketogenesis J. Denis McGarry, Carlos Robles-Valdes, and Daniel W. Foster
p
REVIOUS S T U D I E S from this laboratory 1 led us to suggest that in the rat the development of the ketotic state involves metabolic adaptations in two organ systems, adipose tissue and liver, which in turn are governed largely by two hormones, insulin and glucagon. The following sequence of events was proposed. At the level of adipose tissue accelerated lipolysis, triggered by insulin deficiency, results in mobilization of fatty acids to the liver. At the latter site glucagon excess induces an activation of the carnitine acyltransferase system of enzymes with the result that the fatty acids taken up are efficiently oxidized with the production of acetoacetic and/3-hydroxybutyric acids. Studies with humans carried out by other groups 2 4 are consistent with the above formulation. An important factor in the regulation of hepatic fatty acid oxidation, and thus ketogenesis, was shown to be the tissue concentration of carnitine (a substrate for the carnitine acyltransferase I reaction) which correlated directly with ketogenic capacity over a wide range of nutritional and hormonal manipulations. 5 Moreover, addition of carnitine to the medium perfusing livers from fed, nonketotic rats produced a marked stimulation of the conversion of oleic acid into ketone bodies. 5 To gain further insight into this phenomenon we have now examined the relationship between liver carnitine levels and the regulation of hepatic fatty acid oxidation in the developing rat, since many of the metabolic adaptations that accompany the transition from intrauterine to extrauterine life in this species resemble those seen in adult animals during the transition from the fed (nonketotic) to the fasted (ketotic state). Thus, the primary metabolic fuel of the fetus is glucose derived from maternal blood, 6 liver glycogen is abundant, the ratio of [glucagon]:[insulin] in circulation is lOW, 7 a s is the capacity for fatty acid oxidation in liver, 8'9 and blood ketone levels are normal. 7 In contrast, the neonate receives a high fat diet from maternal milk, 1~ exhibits a high blood [glucagon]:[insulin] ratio, 7 low liver glycogen stores, v a striking increase in hepatic fatty acid oxidation capacity and a marked elevation in blood ketone body concentration. 8,9 As seen from Table 1, the sharp increase in plasma ketones of the neonate, which occurred in synchrony with the switch from a low to a high blood [glucagon]:[insulin] ratio noted by others, was accompanied by a striking elevation of liver carnitine content. Both parameters gradually reverted to normal From the Departments of Internal Medicine and Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Tex. Supported by USPHS Grant A M 18573, Training Grant CA 05200 and a grant from the American Diabetes Association, Dr. McGarry is a recipient of Research Career Development Award 1-KO4-A M0763. Reprint requests should be addressed to J. Denis McGarry, Ph.D., Department of Internal Medicine, The University of Texas Health Science Center at Dallas, Dallas, Tex. 75235. | 1976 by Grune & Stratton, Inc,
Metabolism, Vol. 25, No. 11, Suppl. 1 (November), 1976
1387
1388
MC GARRY, ROBLES-VALDES,
A N D FOSTER
Table 1. Concentrations of Carnitine (Free + Esterified), Glycogen and Ketone Bodies in Various Tissues of Maternal, Fetal, and Neonatal Rats Days Postpartum -1
1
3
8
16
20
23
--
--
Weaned
Plasma Ketones (mM) Babies Mothers .
..
Liver Carmtme (nmol.g
-1
Mothers Milk Carnitine (nmol.m1-1 ) .
--1
2.39
1.64
1.14
0.97
4-0.40
4-0.18
4-0.09
4-0.08
0.15
0.44
0.18
0.25
0.35
4-0.04
4-0.09
4-0.05
4-0.04
4-0.02
0.26 4-0.03
--
--
0.46 4-0.09
)
Babies
bver Glycogen (mg.g
0.22 4-0.05
232
572
4- 22
:k 28
404
544
4- 44
:k 75
--
•
i
409
386
293
27
4- 18
4- 16
532
144
129
82
4- 16
4- 12
271
240
:k 17
4- 22
--
91 =k10
--
--
112
4- 25
122
76
11
4- 8
~
•
--
88
89
7
4- 3
--
)
Babies
93*
Mothers
--
<5
.
.
.
.
.
.
52.6
.
.
.
.
.
.
4- 4.1 Values (means 4- SEM) for
fetal
and
neonatal
tissues
were
obtained
from
pooled
samples
of
4 - 8 litters, and for maternal tissues from 4 to 6 animals. *Taken from Girard et al. 7
during the suckling period. Unlike the situation in ketotic adult rats, however, in which the increased liver carnitine is endogenously derived, that in the neonate is derived largely from maternal milk whose carnitine content was also high on day 1 postpartum and fell continuously thereafter. In experiments not shown, the source of milk carnitine was found to be the maternal liver whose carnitine concentration increased abruptly around the time of parturition and declined between the 3rd and 8th days of nursing. A surprising finding was that despite their high liver carnitine content, the mother rats exhibited little if any ketonemia. That this was not simply due to low plasma free fatty acid levels (0.2-0.3 raM), expected for animals consuming a high carbohydrate diet, was shown by the fact that on perfusion with oleic acid these livers exhibited a low ketogenic capacity. However, unlike the livers from neonatal rats and those from ketotic adult animals which, in addition to being enriched with carnitine, were also found to be uniformly glycogen depleted, the maternal rat liver contained large quantities of glycogen. We conclude from these studies that maximal activation of the hepatic fatty acid oxidation machinery (carnitine acyltransferase) requires both an increase in carnitine and depletion of the glycogen content of the tissue. Both conditions are met under circumstances of relative or absolute glucagon excess. Two questions remain to be resolved. First, how does the maternal liver acquire its increased content of carnitine under circumstances where presumably neither glucagon excess nor insulin deficiency prevails? Second, does glycogen play a direct role in the regulation of hepatic fatty acid oxidation or does its level simply reflect the concentration of another component of carbohydrate metabolism important for the control of carnitine acyltransferase?
GLUCAGON AND KETOGENESIS
1389
REFERENCES 1. McGarry JD, Wright PH, Foster DW: Hormonal control of ketogenesis. Rapid activation of hepatic ketogenic capacity in fed rats by anti-insulin serum and glucagon. J Clin Invest 55:1202-1209, 1975 2. Liljenquist JE, Bomboy JD, Lewis SB, Sinclair-Smith BC, Felts PW, Lacy WW, Crofford OB, Liddle GW: Effects of glucagon on lipolysis and ketogenesis in normal and diabetic men. J Clin Invest 53:190-197, 1974 3. Gerich JE, Lorenzi M, Bier DM, Schneider V, Tsalikian E, Karam JH, Forsham PH: Prevention of human diabetic ketoacidosis by somatostatin. Evidence for an essential role of glucagon. N Engl J Med 292:985-989, 1975 4. Schade DS, Eaton RP: Glucagon regulation of plasma ketone body concentration in human diabetes. J Clin Invest 56:1340-1344, 1975 5. McGarry JD, Robles-Valdes C, Foster DW: Role of carnitine in hepatic ketogenesis. Proc Natl Acad Sci USA 72:4385 4388, 1975
6. Bailey E, Lockwood EA: Some aspects of fatty acid oxidation and ketone body formation and utilization during development of the rat. Enzyme 15:239-253, 1973 7. Girard JR, Cuendet GS, Marliss EB, Kervran A, Rieutort M, Assan R: Fuels, hormones and liver metabolism at term and during the early postnatal period in the rat. J Clin Invest 52:3190-3200. 1973 8. Augenfeld J, Fritz 1B: Carnitine palmitoyltransferase activity and fatty acid oxidation by livers from fetal and neonatal rats. Can J Biochem 48:288-294, 1970 9. Lockwood EA, Bailey E: Fatty acid utilization during development of the rat. Biochem J 120:49-54, 1970 10. Dyrnsza HA, Czajka DM, Miller SA: Influence of artificial diet on weight gain and body composition of the neonatal rat. J Nutrition 84: 100 106, 1964
Discussion Dr. Unger: With respect to the studies of Drs. Sherwin and Felig that the purpose of glucagon is not to cause hyperglycemia--it is to prevent hypoglycemia. Down-regulation of glucagon receptors during the unnatural constant rate experimental infusion of glucagon is a teleologically reasonable defense against hyperglycemia and speaks for nature's ability to defend itself against inappropriate secretion of hormones. Second, during the suppression of IRG, the residual "background" levels of IRG cannot be assumed without proper evidence to be biologically inactive. Perhaps we are underestimating the importance of the residual IRG that always remains during suppression with somatostatin and glucose. Finally, I think Dr. Felig's group and our group are not really as far apart as it seems. Dr. Felig feels, as do we, that glucagon is important in preventing hypoglycemia in the fasting state and during protein-induced insulin secretion. We agree with him that glucagon is not involved in the disposal of an exogenous glucose load, that it is only a hormone of glucose production, and that insulin is the hormone of glucose utilization. So, if glucagon is responsible for a rate of glucose production of about 70 rag/minute basal state, during the glucose tolerance test, when 500-800 rng/minute may be entering the circulation, an effect of glucagon would, if present, be far too trivial to discern. In fact, Dr. Ismet Aydin has infused glucose at a rate of 70 rag/rain, which we calculate would approximate the basal contribution of glucagon to glucose production, beginning 90 rain before the administration of 100 g of oral glucose load. The glucose tolerance and insulin levels were not different from those obtained with a saline control infusion during an oral glucose tolerance test in the same patients. Dr. Gerich: I would just like to comment on Dr. Sherwin's slides in the dog, because we have done something very similar in man. We studied diabetic subjects in whom we had infused with insulin and maintained their blood sugars at about 100 rag/100 ml. Then we stopped the insulin and infused somatostatin for 18 hr. Now this contrasted with their study of 7 hr, but we found something very similar--that after an initial fall in blood sugar down between 60% and 70 rag/100 ml, there was a progressive rise. That rise averaged about 150, which was about the same value as in the dogs. But then they plateaued for about 4 hr and then when we stopped the somatostatin,
1390
MC GARRY, ROBLES-VALDES, AND FOSTER
instead of a fall, as Dr. Sherwin found in the dogs, there was a rise in the blood sugar, up to over 300 m g / 1 0 0 ml. Now, I think perhaps the difference would be that you are using normal dogs which, when the somatostatin was stopped, exhibited some degree of hyperglycemia. But then they had a large burst of insulin and that could explain the fall. But, basically, the results are very similar and they would suggest that you can get m o d e s t hyperglycemia despite glucagon deficiency. N o w we had background I R G so we don't know whether we blocked glucagon completely or not. On the other hand, it does show that 18 hr after insulin you really get only a mild form of diabetes, which suggests that glucagon does modify the degree of hyperglycemia. Dr. Miiller: I do not agree entirely with the interpretation of Dr. Felig. I think that glucagon is important in more than one situation, but I wonder whether your interpretation is really correct when you take normal subjects and there I wonder that you do not relate its importance to insulin. If you give glucagon and you show that insulin goes up in the periphery, surely it must have risen in the portal vein about twice as much, if we accept 5 0 ~ extraction. It is possible to challenge your interpretation because you do not control those insulin levels in the portal area. Dr. Felig: Roger U n g e r and I agree on m a n y more points than we differ. We both agree that glucagon is not an important h o r m o n e in altering glucose disposal. T h e n what's the significance to the loss of alpha cell suppression by glucose? M u c h attention is now being paid to the fact that in diabetes there is a loss o f alpha cell response to glucose suppression. If, in fact, we all agree that glucagon is not important in the disposal of glucose, then 1 think those observations are really without clear physiologic counterpart, and 1 think that would be a major distinction between Dr. U n g e r and me. I believe that failure of suppression of glucagon by glucose, whether it's due to insulin deficiency or a basic part of the diabetic syndrome, is not very important. In terms of the response to protein, both of us would say that this is the circumstance in which glucagon is playing a role in the diabetic. It m a y well be, as John Gerich's work shows, that if you get a burst of glucagon from a protein meal you are raising the blood glucose level and you can't dispose of it. Thus you can get a stepwise buildup of blood glucose ingestion. However, this effect is still dependent on antecedent insulin lack. We have recently published (Warren, J, et al, J Clin Invest, 1976) evidence that the e n d o g e n o u s hyperglucagonemia of a protein meal as in the case of exogenous glucagon has only an evanescent effect on splanchnic glucose output. The persistence of the hyperglycemia thus depends on insulin lack. As far as Walter Mtiller's c o m m e n t s with regard to portal insulin concentration, we obviously have not measured them. The small increase in insulin occurred before the glucose was administered. The glucose level in normal individuals tended to fall back before the glucose was administered. There were no differences in insulin in the two groups. You would have to speculate that in this special circumstance there is a high portal level that we can't detect. It may be true, but it seems unlikely. Dr. Raskin." Small differences in peripheral insulin levels m a y not appear to be important, but, indeed, a change from 8 u U / m l to 12 u U / m l in the peripheral plasma is important. We've repeated some of Dr. Sherwin's experiments and infused glucagon 3 m g / k g / m i n for 3 hr in normal subjects and found exactly what he d i d - - t h e blood sugar goes up a bit. But if one continues the glucagon infusion for 3 hr, and then stops the glucagon infusion, the insulin levels fall. But despite this the blood sugar falls right along with it, evidence that the blood sugar was being maintained by the infused glucagon. Dr. Unger: The failure of the suppression of I R G by a glucose load m a y be important only as a special test of A-cell function. Glucose loads are not normal meals. With normal meals, the plasma I R G rises both in nondiabetic and diabetic persons, and the lowest I R G level of the day is just before breakfast. It is the mealtime rise in I R G that Jack Gerich first showed could be blocked with somatostatin and it is this that m a y be a significant factor in the postprandial hyperglycemia and the ultimate target o f glucagon suppressive therapy. Suppression during a glucose meal m a y be no more than a good test of A-cell function and otherwise is probably devoid of physiologic importance, as Dr. Felig pointed out. Dr. Fo~: I would like to c o m m e n t that we can support Dr. Sperling's findings completely. In rats, just before birth, glucagon is low, insulin is high, hepatic glycogen reaches about 10~. At birth this suppression is completely reversed, glucagon rises, insulin goes down, hepatic glycogen disappears. Dr. Gerich: We were aware of Denis M c G a r r y ' s work and his theory. We have reported that
G L U C A G O N AND KETOGENESIS
1391
somatostatin can prevent the rise both in blood glucose and in /3-hydroxybutyrate levels in juvenile diabetics after stopping their insulin infusion. If glucagon is infused along with the somatostatin, the rise in /3-hydroxybutyrate occurs. Since then we've asked the question, does glucagon also play a role in the maintaining of ketogenesis after insulin withdrawal. We infused insulin for 14 hr after withdrawing patients from their long-acting insulin, then stopped the infusion, allowing them to get hyperglycemic and hyperketonemic for 6 hr and then began a somatostatin infusion. The glucose levels fall progressively, so after 10 hr of insulin deficiency they were much lower, but the r levels did not actually fall, but they did not continue to rise as in the control experiments. So, this data would suggest that maintenance of absolute or relative hyperglucagonemia in the virtual absence of insulin plays some role in maintaining ketogenesis. Dr. Fob: Another possible way of studying the relationship between insulin and glucagon is to measure the secretion of the latter in animals with endogenous hyperinsulinism. J. C. Dunbar, M. Walsh, and I isolated pancreatic islets from hamsters with hypoglycemia and hyperinsulinism due to a transplantable insulinoma and found that they secrete less glucagon than the islets of control animals, suggesting that they may have been inhibited by the high circulating insulin levels.
Glucose in the medium
Glucagon
Insulin
pg/islet/2 hr
#U/islet/2 hr
rag/100 ml
ContrM
Tumor
Control
30
323 • 33
108 d: 22 +
173 • 23
100
229 • 43
138 :~_ 23 +
334 • 37
132 • 20*
300
203 • 32
117 • 25 +
952 • 74
588 • 37*
* = p < 0.05. + =p <0.01. :k = SEM.
Tumor 27 •
10 +
p
REVIOUS S T U D I E S from this laboratory 1 led us to suggest that in the rat the development of the ketotic state involves metabolic adaptations in two organ systems, adipose tissue and liver, which in turn are governed largely by two hormones, insulin and glucagon. The following sequence of events was proposed. At the level of adipose tissue accelerated lipolysis, triggered by insulin deficiency, results in mobilization of fatty acids to the liver. At the latter site glucagon excess induces an activation of the carnitine acyltransferase system of enzymes with the result that the fatty acids taken up are efficiently oxidized with the production of acetoacetic and/3-hydroxybutyric acids. Studies with humans carried out by other groups 2 4 are consistent with the above formulation. An important factor in the regulation of hepatic fatty acid oxidation, and thus ketogenesis, was shown to be the tissue concentration of carnitine (a substrate for the carnitine acyltransferase I reaction) which correlated directly with ketogenic capacity over a wide range of nutritional and hormonal manipulations. 5 Moreover, addition of carnitine to the medium perfusing livers from fed, nonketotic rats produced a marked stimulation of the conversion of oleic acid into ketone bodies. 5 To gain further insight into this phenomenon we have now examined the relationship between liver carnitine levels and the regulation of hepatic fatty acid oxidation in the developing rat, since many of the metabolic adaptations that accompany the transition from intrauterine to extrauterine life in this species resemble those seen in adult animals during the transition from the fed (nonketotic) to the fasted (ketotic state). Thus, the primary metabolic fuel of the fetus is glucose derived from maternal blood, 6 liver glycogen is abundant, the ratio of [glucagon]:[insulin] in circulation is lOW, 7 a s is the capacity for fatty acid oxidation in liver, 8'9 and blood ketone levels are normal. 7 In contrast, the neonate receives a high fat diet from maternal milk, 1~ exhibits a high blood [glucagon]:[insulin] ratio, 7 low liver glycogen stores, v a striking increase in hepatic fatty acid oxidation capacity and a marked elevation in blood ketone body concentration. 8,9 As seen from Table 1, the sharp increase in plasma ketones of the neonate, which occurred in synchrony with the switch from a low to a high blood [glucagon]:[insulin] ratio noted by others, was accompanied by a striking elevation of liver carnitine content. Both parameters gradually reverted to normal From the Departments of Internal Medicine and Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Tex. Supported by USPHS Grant A M 18573, Training Grant CA 05200 and a grant from the American Diabetes Association, Dr. McGarry is a recipient of Research Career Development Award 1-KO4-A M0763. Reprint requests should be addressed to J. Denis McGarry, Ph.D., Department of Internal Medicine, The University of Texas Health Science Center at Dallas, Dallas, Tex. 75235. | 1976 by Grune & Stratton, Inc,
Metabolism, Vol. 25, No. 11, Suppl. 1 (November), 1976
1387
1388
MC GARRY, ROBLES-VALDES,
A N D FOSTER
Table 1. Concentrations of Carnitine (Free + Esterified), Glycogen and Ketone Bodies in Various Tissues of Maternal, Fetal, and Neonatal Rats Days Postpartum -1
1
3
8
16
20
23
--
--
Weaned
Plasma Ketones (mM) Babies Mothers .
..
Liver Carmtme (nmol.g
-1
Mothers Milk Carnitine (nmol.m1-1 ) .
--1
2.39
1.64
1.14
0.97
4-0.40
4-0.18
4-0.09
4-0.08
0.15
0.44
0.18
0.25
0.35
4-0.04
4-0.09
4-0.05
4-0.04
4-0.02
0.26 4-0.03
--
--
0.46 4-0.09
)
Babies
bver Glycogen (mg.g
0.22 4-0.05
232
572
4- 22
:k 28
404
544
4- 44
:k 75
--
•
i
409
386
293
27
4- 18
4- 16
532
144
129
82
4- 16
4- 12
271
240
:k 17
4- 22
--
91 =k10
--
--
112
4- 25
122
76
11
4- 8
~
•
--
88
89
7
4- 3
--
)
Babies
93*
Mothers
--
<5
.
.
.
.
.
.
52.6
.
.
.
.
.
.
4- 4.1 Values (means 4- SEM) for
fetal
and
neonatal
tissues
were
obtained
from
pooled
samples
of
4 - 8 litters, and for maternal tissues from 4 to 6 animals. *Taken from Girard et al. 7
during the suckling period. Unlike the situation in ketotic adult rats, however, in which the increased liver carnitine is endogenously derived, that in the neonate is derived largely from maternal milk whose carnitine content was also high on day 1 postpartum and fell continuously thereafter. In experiments not shown, the source of milk carnitine was found to be the maternal liver whose carnitine concentration increased abruptly around the time of parturition and declined between the 3rd and 8th days of nursing. A surprising finding was that despite their high liver carnitine content, the mother rats exhibited little if any ketonemia. That this was not simply due to low plasma free fatty acid levels (0.2-0.3 raM), expected for animals consuming a high carbohydrate diet, was shown by the fact that on perfusion with oleic acid these livers exhibited a low ketogenic capacity. However, unlike the livers from neonatal rats and those from ketotic adult animals which, in addition to being enriched with carnitine, were also found to be uniformly glycogen depleted, the maternal rat liver contained large quantities of glycogen. We conclude from these studies that maximal activation of the hepatic fatty acid oxidation machinery (carnitine acyltransferase) requires both an increase in carnitine and depletion of the glycogen content of the tissue. Both conditions are met under circumstances of relative or absolute glucagon excess. Two questions remain to be resolved. First, how does the maternal liver acquire its increased content of carnitine under circumstances where presumably neither glucagon excess nor insulin deficiency prevails? Second, does glycogen play a direct role in the regulation of hepatic fatty acid oxidation or does its level simply reflect the concentration of another component of carbohydrate metabolism important for the control of carnitine acyltransferase?
GLUCAGON AND KETOGENESIS
1389
REFERENCES 1. McGarry JD, Wright PH, Foster DW: Hormonal control of ketogenesis. Rapid activation of hepatic ketogenic capacity in fed rats by anti-insulin serum and glucagon. J Clin Invest 55:1202-1209, 1975 2. Liljenquist JE, Bomboy JD, Lewis SB, Sinclair-Smith BC, Felts PW, Lacy WW, Crofford OB, Liddle GW: Effects of glucagon on lipolysis and ketogenesis in normal and diabetic men. J Clin Invest 53:190-197, 1974 3. Gerich JE, Lorenzi M, Bier DM, Schneider V, Tsalikian E, Karam JH, Forsham PH: Prevention of human diabetic ketoacidosis by somatostatin. Evidence for an essential role of glucagon. N Engl J Med 292:985-989, 1975 4. Schade DS, Eaton RP: Glucagon regulation of plasma ketone body concentration in human diabetes. J Clin Invest 56:1340-1344, 1975 5. McGarry JD, Robles-Valdes C, Foster DW: Role of carnitine in hepatic ketogenesis. Proc Natl Acad Sci USA 72:4385 4388, 1975
6. Bailey E, Lockwood EA: Some aspects of fatty acid oxidation and ketone body formation and utilization during development of the rat. Enzyme 15:239-253, 1973 7. Girard JR, Cuendet GS, Marliss EB, Kervran A, Rieutort M, Assan R: Fuels, hormones and liver metabolism at term and during the early postnatal period in the rat. J Clin Invest 52:3190-3200. 1973 8. Augenfeld J, Fritz 1B: Carnitine palmitoyltransferase activity and fatty acid oxidation by livers from fetal and neonatal rats. Can J Biochem 48:288-294, 1970 9. Lockwood EA, Bailey E: Fatty acid utilization during development of the rat. Biochem J 120:49-54, 1970 10. Dyrnsza HA, Czajka DM, Miller SA: Influence of artificial diet on weight gain and body composition of the neonatal rat. J Nutrition 84: 100 106, 1964
Discussion Dr. Unger: With respect to the studies of Drs. Sherwin and Felig that the purpose of glucagon is not to cause hyperglycemia--it is to prevent hypoglycemia. Down-regulation of glucagon receptors during the unnatural constant rate experimental infusion of glucagon is a teleologically reasonable defense against hyperglycemia and speaks for nature's ability to defend itself against inappropriate secretion of hormones. Second, during the suppression of IRG, the residual "background" levels of IRG cannot be assumed without proper evidence to be biologically inactive. Perhaps we are underestimating the importance of the residual IRG that always remains during suppression with somatostatin and glucose. Finally, I think Dr. Felig's group and our group are not really as far apart as it seems. Dr. Felig feels, as do we, that glucagon is important in preventing hypoglycemia in the fasting state and during protein-induced insulin secretion. We agree with him that glucagon is not involved in the disposal of an exogenous glucose load, that it is only a hormone of glucose production, and that insulin is the hormone of glucose utilization. So, if glucagon is responsible for a rate of glucose production of about 70 rag/minute basal state, during the glucose tolerance test, when 500-800 rng/minute may be entering the circulation, an effect of glucagon would, if present, be far too trivial to discern. In fact, Dr. Ismet Aydin has infused glucose at a rate of 70 rag/rain, which we calculate would approximate the basal contribution of glucagon to glucose production, beginning 90 rain before the administration of 100 g of oral glucose load. The glucose tolerance and insulin levels were not different from those obtained with a saline control infusion during an oral glucose tolerance test in the same patients. Dr. Gerich: I would just like to comment on Dr. Sherwin's slides in the dog, because we have done something very similar in man. We studied diabetic subjects in whom we had infused with insulin and maintained their blood sugars at about 100 rag/100 ml. Then we stopped the insulin and infused somatostatin for 18 hr. Now this contrasted with their study of 7 hr, but we found something very similar--that after an initial fall in blood sugar down between 60% and 70 rag/100 ml, there was a progressive rise. That rise averaged about 150, which was about the same value as in the dogs. But then they plateaued for about 4 hr and then when we stopped the somatostatin,
1390
MC GARRY, ROBLES-VALDES, AND FOSTER
instead of a fall, as Dr. Sherwin found in the dogs, there was a rise in the blood sugar, up to over 300 m g / 1 0 0 ml. Now, I think perhaps the difference would be that you are using normal dogs which, when the somatostatin was stopped, exhibited some degree of hyperglycemia. But then they had a large burst of insulin and that could explain the fall. But, basically, the results are very similar and they would suggest that you can get m o d e s t hyperglycemia despite glucagon deficiency. N o w we had background I R G so we don't know whether we blocked glucagon completely or not. On the other hand, it does show that 18 hr after insulin you really get only a mild form of diabetes, which suggests that glucagon does modify the degree of hyperglycemia. Dr. Miiller: I do not agree entirely with the interpretation of Dr. Felig. I think that glucagon is important in more than one situation, but I wonder whether your interpretation is really correct when you take normal subjects and there I wonder that you do not relate its importance to insulin. If you give glucagon and you show that insulin goes up in the periphery, surely it must have risen in the portal vein about twice as much, if we accept 5 0 ~ extraction. It is possible to challenge your interpretation because you do not control those insulin levels in the portal area. Dr. Felig: Roger U n g e r and I agree on m a n y more points than we differ. We both agree that glucagon is not an important h o r m o n e in altering glucose disposal. T h e n what's the significance to the loss of alpha cell suppression by glucose? M u c h attention is now being paid to the fact that in diabetes there is a loss o f alpha cell response to glucose suppression. If, in fact, we all agree that glucagon is not important in the disposal of glucose, then 1 think those observations are really without clear physiologic counterpart, and 1 think that would be a major distinction between Dr. U n g e r and me. I believe that failure of suppression of glucagon by glucose, whether it's due to insulin deficiency or a basic part of the diabetic syndrome, is not very important. In terms of the response to protein, both of us would say that this is the circumstance in which glucagon is playing a role in the diabetic. It m a y well be, as John Gerich's work shows, that if you get a burst of glucagon from a protein meal you are raising the blood glucose level and you can't dispose of it. Thus you can get a stepwise buildup of blood glucose ingestion. However, this effect is still dependent on antecedent insulin lack. We have recently published (Warren, J, et al, J Clin Invest, 1976) evidence that the e n d o g e n o u s hyperglucagonemia of a protein meal as in the case of exogenous glucagon has only an evanescent effect on splanchnic glucose output. The persistence of the hyperglycemia thus depends on insulin lack. As far as Walter Mtiller's c o m m e n t s with regard to portal insulin concentration, we obviously have not measured them. The small increase in insulin occurred before the glucose was administered. The glucose level in normal individuals tended to fall back before the glucose was administered. There were no differences in insulin in the two groups. You would have to speculate that in this special circumstance there is a high portal level that we can't detect. It may be true, but it seems unlikely. Dr. Raskin." Small differences in peripheral insulin levels m a y not appear to be important, but, indeed, a change from 8 u U / m l to 12 u U / m l in the peripheral plasma is important. We've repeated some of Dr. Sherwin's experiments and infused glucagon 3 m g / k g / m i n for 3 hr in normal subjects and found exactly what he d i d - - t h e blood sugar goes up a bit. But if one continues the glucagon infusion for 3 hr, and then stops the glucagon infusion, the insulin levels fall. But despite this the blood sugar falls right along with it, evidence that the blood sugar was being maintained by the infused glucagon. Dr. Unger: The failure of the suppression of I R G by a glucose load m a y be important only as a special test of A-cell function. Glucose loads are not normal meals. With normal meals, the plasma I R G rises both in nondiabetic and diabetic persons, and the lowest I R G level of the day is just before breakfast. It is the mealtime rise in I R G that Jack Gerich first showed could be blocked with somatostatin and it is this that m a y be a significant factor in the postprandial hyperglycemia and the ultimate target o f glucagon suppressive therapy. Suppression during a glucose meal m a y be no more than a good test of A-cell function and otherwise is probably devoid of physiologic importance, as Dr. Felig pointed out. Dr. Fo~: I would like to c o m m e n t that we can support Dr. Sperling's findings completely. In rats, just before birth, glucagon is low, insulin is high, hepatic glycogen reaches about 10~. At birth this suppression is completely reversed, glucagon rises, insulin goes down, hepatic glycogen disappears. Dr. Gerich: We were aware of Denis M c G a r r y ' s work and his theory. We have reported that
G L U C A G O N AND KETOGENESIS
1391
somatostatin can prevent the rise both in blood glucose and in /3-hydroxybutyrate levels in juvenile diabetics after stopping their insulin infusion. If glucagon is infused along with the somatostatin, the rise in /3-hydroxybutyrate occurs. Since then we've asked the question, does glucagon also play a role in the maintaining of ketogenesis after insulin withdrawal. We infused insulin for 14 hr after withdrawing patients from their long-acting insulin, then stopped the infusion, allowing them to get hyperglycemic and hyperketonemic for 6 hr and then began a somatostatin infusion. The glucose levels fall progressively, so after 10 hr of insulin deficiency they were much lower, but the r levels did not actually fall, but they did not continue to rise as in the control experiments. So, this data would suggest that maintenance of absolute or relative hyperglucagonemia in the virtual absence of insulin plays some role in maintaining ketogenesis. Dr. Fob: Another possible way of studying the relationship between insulin and glucagon is to measure the secretion of the latter in animals with endogenous hyperinsulinism. J. C. Dunbar, M. Walsh, and I isolated pancreatic islets from hamsters with hypoglycemia and hyperinsulinism due to a transplantable insulinoma and found that they secrete less glucagon than the islets of control animals, suggesting that they may have been inhibited by the high circulating insulin levels.
Glucose in the medium
Glucagon
Insulin
pg/islet/2 hr
#U/islet/2 hr
rag/100 ml
ContrM
Tumor
Control
30
323 • 33
108 d: 22 +
173 • 23
100
229 • 43
138 :~_ 23 +
334 • 37
132 • 20*
300
203 • 32
117 • 25 +
952 • 74
588 • 37*
* = p < 0.05. + =p <0.01. :k = SEM.
Tumor 27 •
10 +