Metabolism
-
Clinical and Experimental VOL. XXV, NO. 3 PRELIMINARY
MARCH 1976
REPORT
Altered Insulin and Glucagon Secretion in Treated Genetic Hyperlipemia: A Mechanism of Therapy? R. Philip Eaton, Roger Oase, and David S. Schade The influence of Halofenate therapy on insulin and glucagon secretion was examined in the Zucker rat with genetic endogenous hyperlipemia. Coincident with the lipid lowering ethcts of Halofenate, the net change in the basal bihormonal axis favored glueagon, with the I/G molar ratio (Insulin/Glucagon) decreasing from 2.72 f 0.53 to 0.96 f 0.20 during treatment with this drug. Following arginine stimulation the I/G ratio remained reduced at 0.87 f 0.13 in Halofenah treated
animals, contmsting with the statistically greater ratio of 2.5 f 0.55 in control animals. The Halofenate induced state of reduced insuliruglucagon was associated with hypolipemia, postarginine hyperglycemia, and hyperketonemia,-three metabolic pammeters characteristic of glucagon excess relative to insulin. It is suggested that the lipid-lowering action of Halofenate in genetic hyperlipemia may reflect the altered bihormonal axis induced by the drug.
E HAVE PREVIOUSLY SUGGESTED’ that some forms of acquired and genetic endogenous hyperlipemia (type IV) may be maintained by a reduction in the “lipid-lowering” actions of glucagon relative to the “lipidelevating” actions of insulin. The present study was conducted to define the bihormonal axis i’f a genetic animal model of human endogenous hyperlipemia: and to explore a potential alteration of this axis as the mechanism of action of the new hypolipemic drug Halofenate (MK-185*). These observations
*2-acetamicoethyl (p-chlorophenyl, m-triflurormethylphenoxy) acetate. From the Department of Medicine, University of New Mexico School of Medicine, Albuquerque. N. Mex. Received for publication April 21.1975. Supported by Career Development A ward 1 K04 35843, a grant from the Merck Sharp and Dohme Company. and a grant from the KROC Foundation. Reprint requests should be addressed to Dr. R. Philip Eaton, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, N. Mex. 87131. 0 1976 by Grune & Stratton, Inc.
Metabo/ism, Vol. 25, No. 3 (March), 1976
245
246
EATON,
OASE,
AND
SCHADE
in a rat model of type IV lipemia support the hypothesis that suppression of insulin concentration in association with normal glucagon levels may be implicated in the hypolipemic action of this drug. MATERIALS
AND METHODS
Studies were performed on 44 pair-fed male Zucker homozygous hyperlipemic rats, weighing 490 f 50 g. Twenty-four animals were utilized for arginine stimulation of hormone secretion, while the remaining twenty animals were used for basal examination of hormone and substrate concentrations. Food consumption (25 f 2 gm/day) was identical in treated and control animals, with mean body weight at the time of sacrifice being statistically indistinguishable (p > 0.5). Fifteen milligrams of Halofenate (provided by Merck and Company, Inc., Rahway, New Jersey, Lot #L599, SSI-OOPOS)was administered daily by stomach tube as a 15 mg/ml aqueous solution of the sodium salt, while control littermate hyperlipemic rats received saline alone. After 10 days of treatment, an arginine tolerance test (ATT) arginine, 0.5 mg/g body weight by intraperitoneal injection) was performed as previously described,3 to evaluate simultaneous aminogenic insulin and glucagon secretion. The ATT was performed following a 48-hr fast with ad lib excess to saline, and 18 hr following the last dosage of Halofenate or saline. At each time of observation, following arginine injection, three animals in both the control and Halofenate treated groups were decapitated, and the blood collected with heparin at 5°C. Plasma samples were obtained at 0, 30, 60, and 90 min postarginine, and assayed for glucose, insulin, glucagon, and alpha-amino nitrogen as previously described in rats.3’4 The determinations of basal fasted levels of hormones and substrates were performed on groups of animals separate from those utilized in the arginine tolerance test. In these animals, in addition to the assay of insulin, glucagon, and glucose, we also measured triglyceride, cholesterol, free fatty acid (FFA), and very low density lipoprotein (VLDL-protein) as previously described by Eaton et al. in rats.3’4 Beta-hydroxybutyrate (BOH) and acetoacetate (AcAc) were assayed enzymatically,’ and plasma Halofenate by the method of Barrett and Thorpe6 adapted from the assay of Clofibrate. Addition of Halofenate in vitro (200 pg/ml) did not interfere with the assay of insulin, glucagon, BOH, or AcAc. All data are expressed as the mean f SEM of each group. RESULTS
Basal plasma Halofenate concentration averaged 160 f 15 rg/ml in the drug treated animals, approximating the levels desired in human therapeutic investigation.6 In response to Halofenate therapy, basal plasma triglyceride concentration was reduced 44% and plasma cholesterol SO%, in association with a reduction in VLDL-protein of 40% (see Table 1). Hormone Response
Basal plasma insulin concentration averaged 78 A 8 pU/ml in ten hyperlipemic control rats, and was significantly reduced in ten Halofenate-treated Table 1.
Effect of Halofenate Treatment on Plasma lipid Parameters in 48 Hr pasted Hvaerliaemic Rats”
Parameter Triglyceride Cholesterol Free Fatty
Acids (pEq/L) (rg/ml)
Acetoacetote
(pmol/L)
Betahydroxybutyrote
48-hr
represent
fast with
f
50
132
f
15
56+
1064
f
41 12
176+
(pmol/L) mean
f
SEM
ad lib access to saline
of four and
Holofenate-treated
322
(mg/dl)
VLDL-protein
‘Values
Control
(mg/dl)
1900
f
5700
f
to seven
water.
100 600 rats in each
Change
125 zk 25
group
-61%
5
- 50%
916zt
112
-10%
105+
8
- 40%
2900
zt 100
+ 50%
6500
f
300
evaluated
+140/6 at
8:OO a.m.
following
a
INSULIN
AND GLUCAGON
247
SECRETION
HALCFENATE LIPEMIC RAT
LIPEMIC RAT ARG-TT . ux-
RX AFK-TT
PL‘%MA rucw%N
%o-
Fig. 1. Glucagon, insulin, and glucose response to arginine injection in the genetic hyperlipemic rat, with and without halofenate administmtion. Mean f SEM of three animals at each time point.
rats to 43 f 5 pU/ml ( p < 0.001). In contrast, the basal plasma glucagon concentration of 653 f 103 pg/ml in the ten control hyperlipemic rats was not significantly different (803 f 105 pg/ml) in Halofenate-treated animals (p > 0.1). Thus, the basal insulin:glucagon molar ratio (I/G) was significantly lower (p < 0.005) in the Halofenate treated rats (0.96 f 0.20) than in the salinetreated animals (2.72 + 0.53). Arginine injection resulted in a prompt rise in plasma alpha-amino nitrogen concentration of 2.0 + 0.2 PM/ml above baseline, reflecting the rise in plasma arginine, and was not influenced by Halofenate treatment. As shown in Fig. 1, in the saline treated hyperlipemic animals, arginine injection was associated with a gradual reduction in concentration of plasma insulin (minimum = 42 + 8 PI-l/ml) and glucagon (minimum = 380 f 50 pg/ml). This reduction in hormone concentration was unexpected, since Sprague-Dawley rats’ have been shown to increase insulin and glucagon secretion in response to this aminoacid. When arginine was injected into Halofenate treated rats, a striking augmentation of the hormone response was observed (Fig. 1). While plasma insulin concentration now rose steadily to a maximum of 47 f 6 rU/ml (p < O.OOl), plasma glucagon concentration rose to levels of 1040 f 200 pg/ml by
248
EATON,
OASE, AND SCHADE
30 min postarginine injection ( p < 0.001). At the peak of glucagon secretion, the I/G molar ratio was 2.5 f 0.55 in saline treated lipemic rats, in contrast to a reduced I/G ratio in the Halofenate-treated rats of 0.87 + 0.13 (p < 0.025). Substrate Response
Though basal plasma glucose concentration was only minimally altered by Halofenate treatment (Saline = 104 f 6 mg/lOO ml; Halofenate = 89 + 4 mg/ 100 ml p < 0.05), the postarginine integrated rise in plasma glucose was increased by 47% with drug treatment (Saline = 2520 mg/dl-min; Halofenate = 3705 mg/dl-min, p < 0.005). While basal FFA levels were not significantly altered by the drug (Saline = 1064 + 41 rEq/L; Halofenate = 916 f 112 &Eq/L, p > 0.25), basal total ketone concentration (AcAc + BOH) increased by 24% with a rise of approximately 1000 rmol/L in both AcAc and BOH (p < 0.005) (Table 1). DISCUSSION
The present study was designed to examine the effect of a lipid-lowering agent (Halofenate), in an animal model of human type IV endogenous hyperlipemia as characterized by Schonfeld et al.? The potential role of an alteration in the concentration of insulin and glucagon as a basis for the hypolipemic action of the drug was explored. Our studies demonstrate that Halofenate treatment results in a reduction in the insulin/glucagon molar ratio in both the basal state, and during the ATT, demonstrating drug augmented glucagon secretion relative to insulin secretion in the drug treated hyperlipemic rat. Insulin deficiency results in a marked depression in hepatic triglyceride production and secretion,8 and lipoprotein formation is severely impaired in the liver of the alloxan diabetic rat’ consistent with previous reports that hepatic protein synthesis is an insulin-dependent process.” The effects of glucagon are antagonistic to those of insulin, with glucagon excess depressing triglyceride synthesis” and inhibiting protein formation in the liver.i2 If a true state of relative glucagon excess is induced by Halofenate administration, then the hyperglycemic and ketogenic actions of glucagoni3 might also be observed. Though the data are limited, the 70% greater integrated glucose response to arginine and 24% increase in basal ketones seen following Halofenate administration could reflect such a state of glucagon excess. In man, a similar reduction in arginine-stimulated insulin secretion with potentiation of glucagon secretion has also been reported with the structurally related compound, clofibrate,i4 with a similar reduction in basal endogenous plasma lipids. Extrapolation to the human response to Halofenate must be guarded, since the dose administered, tissue distribution, and biologic effects may be different in human hyperlipemia. Nevertheless, our data suggest that a portion of the hypolipemic action of Halofenate may be mediated by alterations in insulin and glucagon release. REFERENCES 1. Eaton RP, Schade DS, Conway M: Decreased glucagon activity: A mechanism for
both genetic and acquired endogenous lipemia. Lancet 11:1545, 1974
hyper-
INSULIN
AND GLUCAGON
SECRETION
2. Schonfeld G, Felski C, Howald MA: Characterization of the plasma lipoproteins of the genetically obese hyperlipoproteinemic zucker fatty rat. J Lipid Res 15:457, 1974 3. Eaton RP: Effect of clofibrate on arginineinduced insulin and glucagon secretion. Metabolism 22:763, 1973 4. Eaton RP: Glucagon secretion and activity in the cobalt chloride treated rat. Am J Physiol 225~67, 1973 5. Mellanby J, Williamson DH: Acetoacetate, in Bergmeyer HU (ed): Methods in Enzymatic Analysis. New York, Academic Press, 1965, p 454 6. Barrett AM, Thorpe JM: Studies on the mode of action of clofibrate. Br J Pharmacol 32:381, 1968 7. Sirtori C, Hurwitz A, Sabih K, Azarnoff DL: Clinical evaluation of MK-185: A new hypolipidemic drug. Lipids 7:96, 1972 8. Heimberg MVH, Brown TO: Hepatic lipid metabolism in experimental diabetes. Biochim Biophys Acta 137:434, 1967
249
9. Eaton RP, Kipnis DM: Etfect of glucose feeding on lipoprotein synthesis in the rat. Am J Physiol 217:1153, 1969 IO. Krahl ME: Functions of insulin and other regulating factors in peptide formation by animal cells. Recent Prog Horm Res 12:199, 1956 I I. Vaughan M, Steinberg D, Pittman R: On the interpretation of studies measuring uptake and esterification of 1-‘4C-palmitate by rat adipose tissue in-vitro. Biochim Biophys Acta 84: 154, 1964 12. Eaton RP: The hypolipemic action of glucagon in experimental endogenous lipemia in the rat. J Lipid Res 14:312, 1973 13. Schade DS, Eaton RP: Modulation of fatty acid metabolism by glucagon in man: I. Effects in normal subjects. Diabetes 24:502, 1975 14. Eaton RP, Schade DS: The effect of clofibrate on arginine stimulated glucagon and insulin secretion in man. Metabolism 23:445, 1974
-
Clinical and Experimental VOL. XXV, NO. 3 PRELIMINARY
MARCH 1976
REPORT
Altered Insulin and Glucagon Secretion in Treated Genetic Hyperlipemia: A Mechanism of Therapy? R. Philip Eaton, Roger Oase, and David S. Schade The influence of Halofenate therapy on insulin and glucagon secretion was examined in the Zucker rat with genetic endogenous hyperlipemia. Coincident with the lipid lowering ethcts of Halofenate, the net change in the basal bihormonal axis favored glueagon, with the I/G molar ratio (Insulin/Glucagon) decreasing from 2.72 f 0.53 to 0.96 f 0.20 during treatment with this drug. Following arginine stimulation the I/G ratio remained reduced at 0.87 f 0.13 in Halofenah treated
animals, contmsting with the statistically greater ratio of 2.5 f 0.55 in control animals. The Halofenate induced state of reduced insuliruglucagon was associated with hypolipemia, postarginine hyperglycemia, and hyperketonemia,-three metabolic pammeters characteristic of glucagon excess relative to insulin. It is suggested that the lipid-lowering action of Halofenate in genetic hyperlipemia may reflect the altered bihormonal axis induced by the drug.
E HAVE PREVIOUSLY SUGGESTED’ that some forms of acquired and genetic endogenous hyperlipemia (type IV) may be maintained by a reduction in the “lipid-lowering” actions of glucagon relative to the “lipidelevating” actions of insulin. The present study was conducted to define the bihormonal axis i’f a genetic animal model of human endogenous hyperlipemia: and to explore a potential alteration of this axis as the mechanism of action of the new hypolipemic drug Halofenate (MK-185*). These observations
*2-acetamicoethyl (p-chlorophenyl, m-triflurormethylphenoxy) acetate. From the Department of Medicine, University of New Mexico School of Medicine, Albuquerque. N. Mex. Received for publication April 21.1975. Supported by Career Development A ward 1 K04 35843, a grant from the Merck Sharp and Dohme Company. and a grant from the KROC Foundation. Reprint requests should be addressed to Dr. R. Philip Eaton, Department of Medicine, University of New Mexico School of Medicine, Albuquerque, N. Mex. 87131. 0 1976 by Grune & Stratton, Inc.
Metabo/ism, Vol. 25, No. 3 (March), 1976
245
246
EATON,
OASE,
AND
SCHADE
in a rat model of type IV lipemia support the hypothesis that suppression of insulin concentration in association with normal glucagon levels may be implicated in the hypolipemic action of this drug. MATERIALS
AND METHODS
Studies were performed on 44 pair-fed male Zucker homozygous hyperlipemic rats, weighing 490 f 50 g. Twenty-four animals were utilized for arginine stimulation of hormone secretion, while the remaining twenty animals were used for basal examination of hormone and substrate concentrations. Food consumption (25 f 2 gm/day) was identical in treated and control animals, with mean body weight at the time of sacrifice being statistically indistinguishable (p > 0.5). Fifteen milligrams of Halofenate (provided by Merck and Company, Inc., Rahway, New Jersey, Lot #L599, SSI-OOPOS)was administered daily by stomach tube as a 15 mg/ml aqueous solution of the sodium salt, while control littermate hyperlipemic rats received saline alone. After 10 days of treatment, an arginine tolerance test (ATT) arginine, 0.5 mg/g body weight by intraperitoneal injection) was performed as previously described,3 to evaluate simultaneous aminogenic insulin and glucagon secretion. The ATT was performed following a 48-hr fast with ad lib excess to saline, and 18 hr following the last dosage of Halofenate or saline. At each time of observation, following arginine injection, three animals in both the control and Halofenate treated groups were decapitated, and the blood collected with heparin at 5°C. Plasma samples were obtained at 0, 30, 60, and 90 min postarginine, and assayed for glucose, insulin, glucagon, and alpha-amino nitrogen as previously described in rats.3’4 The determinations of basal fasted levels of hormones and substrates were performed on groups of animals separate from those utilized in the arginine tolerance test. In these animals, in addition to the assay of insulin, glucagon, and glucose, we also measured triglyceride, cholesterol, free fatty acid (FFA), and very low density lipoprotein (VLDL-protein) as previously described by Eaton et al. in rats.3’4 Beta-hydroxybutyrate (BOH) and acetoacetate (AcAc) were assayed enzymatically,’ and plasma Halofenate by the method of Barrett and Thorpe6 adapted from the assay of Clofibrate. Addition of Halofenate in vitro (200 pg/ml) did not interfere with the assay of insulin, glucagon, BOH, or AcAc. All data are expressed as the mean f SEM of each group. RESULTS
Basal plasma Halofenate concentration averaged 160 f 15 rg/ml in the drug treated animals, approximating the levels desired in human therapeutic investigation.6 In response to Halofenate therapy, basal plasma triglyceride concentration was reduced 44% and plasma cholesterol SO%, in association with a reduction in VLDL-protein of 40% (see Table 1). Hormone Response
Basal plasma insulin concentration averaged 78 A 8 pU/ml in ten hyperlipemic control rats, and was significantly reduced in ten Halofenate-treated Table 1.
Effect of Halofenate Treatment on Plasma lipid Parameters in 48 Hr pasted Hvaerliaemic Rats”
Parameter Triglyceride Cholesterol Free Fatty
Acids (pEq/L) (rg/ml)
Acetoacetote
(pmol/L)
Betahydroxybutyrote
48-hr
represent
fast with
f
50
132
f
15
56+
1064
f
41 12
176+
(pmol/L) mean
f
SEM
ad lib access to saline
of four and
Holofenate-treated
322
(mg/dl)
VLDL-protein
‘Values
Control
(mg/dl)
1900
f
5700
f
to seven
water.
100 600 rats in each
Change
125 zk 25
group
-61%
5
- 50%
916zt
112
-10%
105+
8
- 40%
2900
zt 100
+ 50%
6500
f
300
evaluated
+140/6 at
8:OO a.m.
following
a
INSULIN
AND GLUCAGON
247
SECRETION
HALCFENATE LIPEMIC RAT
LIPEMIC RAT ARG-TT . ux-
RX AFK-TT
PL‘%MA rucw%N
%o-
Fig. 1. Glucagon, insulin, and glucose response to arginine injection in the genetic hyperlipemic rat, with and without halofenate administmtion. Mean f SEM of three animals at each time point.
rats to 43 f 5 pU/ml ( p < 0.001). In contrast, the basal plasma glucagon concentration of 653 f 103 pg/ml in the ten control hyperlipemic rats was not significantly different (803 f 105 pg/ml) in Halofenate-treated animals (p > 0.1). Thus, the basal insulin:glucagon molar ratio (I/G) was significantly lower (p < 0.005) in the Halofenate treated rats (0.96 f 0.20) than in the salinetreated animals (2.72 + 0.53). Arginine injection resulted in a prompt rise in plasma alpha-amino nitrogen concentration of 2.0 + 0.2 PM/ml above baseline, reflecting the rise in plasma arginine, and was not influenced by Halofenate treatment. As shown in Fig. 1, in the saline treated hyperlipemic animals, arginine injection was associated with a gradual reduction in concentration of plasma insulin (minimum = 42 + 8 PI-l/ml) and glucagon (minimum = 380 f 50 pg/ml). This reduction in hormone concentration was unexpected, since Sprague-Dawley rats’ have been shown to increase insulin and glucagon secretion in response to this aminoacid. When arginine was injected into Halofenate treated rats, a striking augmentation of the hormone response was observed (Fig. 1). While plasma insulin concentration now rose steadily to a maximum of 47 f 6 rU/ml (p < O.OOl), plasma glucagon concentration rose to levels of 1040 f 200 pg/ml by
248
EATON,
OASE, AND SCHADE
30 min postarginine injection ( p < 0.001). At the peak of glucagon secretion, the I/G molar ratio was 2.5 f 0.55 in saline treated lipemic rats, in contrast to a reduced I/G ratio in the Halofenate-treated rats of 0.87 + 0.13 (p < 0.025). Substrate Response
Though basal plasma glucose concentration was only minimally altered by Halofenate treatment (Saline = 104 f 6 mg/lOO ml; Halofenate = 89 + 4 mg/ 100 ml p < 0.05), the postarginine integrated rise in plasma glucose was increased by 47% with drug treatment (Saline = 2520 mg/dl-min; Halofenate = 3705 mg/dl-min, p < 0.005). While basal FFA levels were not significantly altered by the drug (Saline = 1064 + 41 rEq/L; Halofenate = 916 f 112 &Eq/L, p > 0.25), basal total ketone concentration (AcAc + BOH) increased by 24% with a rise of approximately 1000 rmol/L in both AcAc and BOH (p < 0.005) (Table 1). DISCUSSION
The present study was designed to examine the effect of a lipid-lowering agent (Halofenate), in an animal model of human type IV endogenous hyperlipemia as characterized by Schonfeld et al.? The potential role of an alteration in the concentration of insulin and glucagon as a basis for the hypolipemic action of the drug was explored. Our studies demonstrate that Halofenate treatment results in a reduction in the insulin/glucagon molar ratio in both the basal state, and during the ATT, demonstrating drug augmented glucagon secretion relative to insulin secretion in the drug treated hyperlipemic rat. Insulin deficiency results in a marked depression in hepatic triglyceride production and secretion,8 and lipoprotein formation is severely impaired in the liver of the alloxan diabetic rat’ consistent with previous reports that hepatic protein synthesis is an insulin-dependent process.” The effects of glucagon are antagonistic to those of insulin, with glucagon excess depressing triglyceride synthesis” and inhibiting protein formation in the liver.i2 If a true state of relative glucagon excess is induced by Halofenate administration, then the hyperglycemic and ketogenic actions of glucagoni3 might also be observed. Though the data are limited, the 70% greater integrated glucose response to arginine and 24% increase in basal ketones seen following Halofenate administration could reflect such a state of glucagon excess. In man, a similar reduction in arginine-stimulated insulin secretion with potentiation of glucagon secretion has also been reported with the structurally related compound, clofibrate,i4 with a similar reduction in basal endogenous plasma lipids. Extrapolation to the human response to Halofenate must be guarded, since the dose administered, tissue distribution, and biologic effects may be different in human hyperlipemia. Nevertheless, our data suggest that a portion of the hypolipemic action of Halofenate may be mediated by alterations in insulin and glucagon release. REFERENCES 1. Eaton RP, Schade DS, Conway M: Decreased glucagon activity: A mechanism for
both genetic and acquired endogenous lipemia. Lancet 11:1545, 1974
hyper-
INSULIN
AND GLUCAGON
SECRETION
2. Schonfeld G, Felski C, Howald MA: Characterization of the plasma lipoproteins of the genetically obese hyperlipoproteinemic zucker fatty rat. J Lipid Res 15:457, 1974 3. Eaton RP: Effect of clofibrate on arginineinduced insulin and glucagon secretion. Metabolism 22:763, 1973 4. Eaton RP: Glucagon secretion and activity in the cobalt chloride treated rat. Am J Physiol 225~67, 1973 5. Mellanby J, Williamson DH: Acetoacetate, in Bergmeyer HU (ed): Methods in Enzymatic Analysis. New York, Academic Press, 1965, p 454 6. Barrett AM, Thorpe JM: Studies on the mode of action of clofibrate. Br J Pharmacol 32:381, 1968 7. Sirtori C, Hurwitz A, Sabih K, Azarnoff DL: Clinical evaluation of MK-185: A new hypolipidemic drug. Lipids 7:96, 1972 8. Heimberg MVH, Brown TO: Hepatic lipid metabolism in experimental diabetes. Biochim Biophys Acta 137:434, 1967
249
9. Eaton RP, Kipnis DM: Etfect of glucose feeding on lipoprotein synthesis in the rat. Am J Physiol 217:1153, 1969 IO. Krahl ME: Functions of insulin and other regulating factors in peptide formation by animal cells. Recent Prog Horm Res 12:199, 1956 I I. Vaughan M, Steinberg D, Pittman R: On the interpretation of studies measuring uptake and esterification of 1-‘4C-palmitate by rat adipose tissue in-vitro. Biochim Biophys Acta 84: 154, 1964 12. Eaton RP: The hypolipemic action of glucagon in experimental endogenous lipemia in the rat. J Lipid Res 14:312, 1973 13. Schade DS, Eaton RP: Modulation of fatty acid metabolism by glucagon in man: I. Effects in normal subjects. Diabetes 24:502, 1975 14. Eaton RP, Schade DS: The effect of clofibrate on arginine stimulated glucagon and insulin secretion in man. Metabolism 23:445, 1974
