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Superactive somatostatin analog decreases plasma glucose and glucagon levels in diabetic rats
Peptides, Vol. 9, pp. 561-565. ©PergamonPress plc, 1988. Printedin the U.S.A.
0196-9781/88$3.00 + .00
Superactive Somatostatin Analog Decreases Plasma Glucose and Glucagon Levels in Diabetic Rats T S U Y O S H I K A R A S H I M A * A N D A N D R E W V. S C H A L L Y * t I
tEndocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center and *Section of Experimental Medicine, Department of Medicine Tulane University School of Medicine, New Orleans, LA 70146 R e c e i v e d 5 S e p t e m b e r 1987 KARASHIMA, T. AND A. V. SCHALLY. Superactive somatostatin analog decreases plasma glucose and glucagon levels in diabetic rats. PEPTIDES 9(3) 561-565, 1988.--The action of the new analog of somatostatin, DPhe-C~s-Tyr-D-Trp-Lys-Val-C3ls-Trp-NH2 (RC-160), on plasma glucagon and glucose levels was evaluated in streptozotocin-diabetic rats. The effect of this analog on the insulin-induced hypoglycemia in diabetic rats was also investigated in order to evaluate the risk of exacerbating hypoglycemia. Administration of analog RC-160, in a dose of 25 /zg/kg b.wt. SC, inhibited plasma glucagon secretion and decreased plasma glucose levels. This effect also occurred when plasma glucagon and glucose levels were first elevated by arginine infusion, 1000 mg/kg/hr for 30 min. Subcutaneous injection of regular insulin, 15 U/kg b.wt., produced hypoglycemia with a progressive increase in glucagon levels. Analog RC-160 completely suppressed the hypoglycemia-induced glucagon release for up to 150 min after injection of the analog or insulin. A greater decrease in the plasma glucose level was observed in the group treated with insulin and the analog than in the group injected only with insulin. These results indicate that somatostatin analog RC-160 can produce a marked and prolonged inhibition of glucagon release and a decrease in the plasma glucose level in diabetic rats. This analog may be useful as an adjunct to insulin in the treatment of diabetic patients, although caution should be exercised, to prevent hypoglycemia when using somatostatin analogs together with insulin. Somatostatin analog
Diabetes mellitus
Hypoglycemia
T H E effectiveness of glucagon suppression by somatostatin-14 and its analogs has been well documented in diabetic patients [3, 4, 6, 7, 11]. When insulin administration is combined with glucagon suppression, induced by somatostatin or its analog, the dosage of insulin can be reduced [3, 4, 10]. This might be of importance clinically because peripheral hyperinsulinemia, which may contribute to the development of subsequent complications of diabetes mellitus, could thus be avoided [13]. In the treatment of patients with a stable condition of diabetes, it might be worthwhile to try injecting a combination of an intermediate type of insulin and a long-acting somatostatin analog, twice a day, before breakfast and supper. Some somatostatin analogs, which have been developed, could be used for this purpose [7, 10, 12]. We recently synthesized a new somatostatin analog, D-PheC~)s-Tyr-D-Trp-Lys-Val-C}s-Trp-NH2 (RC-160), with prolonged and enhanced activities in addition to a high selectivity in inhibiting glucagon secretion [2, 8, 9]. Since this analog could be useful for treatment of diabetes, we evaluated its effects on the fasting and arginine-elevated glucagon and glucose levels in diabetic rats. Somatostatin analogs could be tried, together with insulin, in patients with a long history of diabetes. Some of these
Glucagon
Blood glucose
patients may experience hypoglycemia due to the dysfunction of their counterregulatory system [1,14]. Since somatostatin analogs can suppress the secretion of glucagon, the main counterregulatory hormone, for a prolonged period, it cannot be excluded that they might produce a serious hypoglycemia in diabetics [13]. We therefore investigated whether the analog would augment the insulin-induced hypoglycemia in diabetic rats. METHOD
Materials Adult male Sprague-Dawley rats weighing 270-350 g were used in all the experiments. Animals were allowed standard diet and tap water ad lib, and were maintained under controlled conditions: 12 hr light/12 hr dark schedule, at 24--2°C. Diabetes was induced by a single IP injection of streptozotocin (Upjohn Research Lab., Kalamazoo, MI), 65 mg/kg b.wt., freshly prepared in 0.05 M citrate and physiological saline, pH 4.5, after an 18 hr fast. Only the rats whose fasting plasma glucose level was in excess of 200 mg/dl were used 4-7 days after the injection of streptozotocin. The number of rats in each group was 8-10.
1Requests for reprints should be addressed to Dr. Andrew V. Schally (151), Veterans Administration Medical Center, 1601 Perdido St., New Orleans, LA 70146.
561
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KARASHIMA AND S C H A L L Y
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50 I 0
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I 90
i 120
i 150
180
(min)
FIG. 1. Effect of SC administration of analog RC-160, 25 tzg/kg b.wt., on plasma glucose (A) and glucagon (B) levels in diabetic rats. The levels are expressed as a percentage of the value at the time 0. Absolute basal values of plasma glucose and glucagon were in the range of 226--368 mg/dl and 105-240 ng/ml, respectively. 0: Control group; 0: Group treated with RC-160; *: p<0.05 (by Duncan's test); **: p<0.01 vs. control (by Duncan's test).
160
140
C_12o 0 n." 100
Pent©barbital (Abbot Laboratories, North Chicago, IL), 50 mg/kg b.wt., IP was used as an anesthetic. The anesthesia was maintained with small additional doses of pent©barbital (10 mg/kg b.wt., hourly). One ml of blood sample was drawn from the jugular vein and replaced with the same volume of physiological saline. Arginine solution was infused into the jugular vein using an infusion pump. In the first experiment, the animals were injected with 25 /zg/kg of analog DPhe-C~,s-Tyr-D-Trp-Lys-Val-C#s-Trp-NH2(RC-160) and the samples were collected every 30 min for 2 hr. In the second experiment 25/xg/kg of RC-160 were injected SC and, 30 rain later, arginine HC1 (Sigma, St. Louis, MO) 1000 mg/kg/hr was infused for 30 min. Blood samples were taken at time 0, and 60 and 90 min after the injection of analog. In the third experiment 25/zg/kg of RC-160 and 15 U/kg b.wt. of regular pork insulin (Squibb-Nov©, Princeton, NJ) were given SC and blood samples were taken every 30 min for 150 rain. All samples were collected in chilled tubes containing 1.2 mg EDTA (Sigma) and 500 U of aprotinin (Sigma). Analog RC-160 was synthesized in our laboratory using solid-phase methods and purified by HPLC [2,8]. The peptide was dissolved with physiological saline containing 0.2% bovine
=on 8 0 0 .=J L9
60
40
,
,
0
30
,
,
60 90 T I M E (rain)
,
120
FIG. 2. Effect of SC administration of analog RC-160, 25 /xg/kg b.wt., on plasma glucagon (A) and glucose (B) levels in diabetic rats, elevated by infusion of arginine, 1000mg/kg/hr for 30 min. The levels are expressed as a percentage of the value at time 0. Absolute basal values of plasma glucagon and glucose were in the range of 120-290 ng/ml and 205-480 mg/dl. O: Control; ©: Group treated with RC160; *: p<0.05 vs. control (by Duncan's test); **:p<0.01 vs. control (by Duncan's test).
SOMATOSTATIN ANALOG AND DIABETIC RAT
563
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500
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120
150
TIME (mln)
FIG. 3. Effect of SC administration of analog RC-160, 25/zg/kg b.wt., on plasma glucagon (A) and glucose (B) levels after SC injection of insulin, 15 U/kg b.wt., in diabetic rats. Plasma glucagon levels are expressed as a percentage of values at time 0. Absolute values of glucagon were in the range of 158--280 ng/ml. Glucose levels are given in absolute values (mg/dl). O: Control; O: Group treated with RC-160; *: p<0.05 (by Duncan's test); **: p<0.01 vs. control (by Duncan's test).
564
KARASHIMA AND SCHALLY
serum albumin. The plasma glucose was determined by a commercial kit using glucose oxidase method (Stanbio, San Antonio, TX). The plasma glucagon was measured by a specific RIA [2,8]. The values of glucose and glucagon, except for glucose levels in the third experiment, were expressed as a percentage of the control [10]. Statistical significance was assessed by Two-Way Analysis of Variance (ANOVA), and by Duncan's multiple range test. RESULTS
As shown in Fig. 1, subcutaneous administration of analog D-Phe-C~'s-Tyr-D-Trp-Lys-Val-C~,s-Trp-NH2 (RC160), at a dose of 25/zg/kg, decreased the basal fasting glucagon level in diabetic rats relative to vehicle-injected controls. By ANOVA, significant differences were found for treatment, F(1,56)=82.1, p<0.0001, and time, F(4,56)=18.39, p<0.0001. The subcutaneous administration of RC-160 also induced a slight but significant reduction in plasma glucose level: two-way ANOVA revealed a significant difference for treatment, F(1,61)=12.92, p<0.001. This difference was largely due to the difference at the 90 min measurement which was significant (o <0.05) by the Duncan's test (Fig. 1). In the second experiment arginine infusion, 1000 mg/kg/hr for 30 min, elevated the fasting plasma glucagon and glucose levels (Fig. 2). A subcutaneous injection of analog RC-160, in a dose of 25/zg/kg, inhibited these increases in glucagon and glucose levels (Fig. 2). Two-way ANOVA for glucagon levels showed significant differences for treatment, F(1,45)=26.93, p<0.0001, and time, F(2,45)=62.94, p<0.0001, and a significant interaction was found between these two factors, F(2,45)=21.34, p<0.0001. By Duncan's test, significant difference was found only at the 60 minute level (p<0.01). For glucose levels, a significant difference was found by ANOVA for treatment, F(1,44)=13.56, p <0.001, and by Duncan's test, a significant difference was detected at the 90 min measurement (p<0.05) (Fig. 2). When the animals were injected SC with 15 U/kg b.wt. of insulin, the plasma glucose was precipitately reduced to hypoglycemia levels, and plasma glucagon levels progressively rose in the third experiment (Fig. 3). Concomitant injection of analog RC-160 completely suppressed the increase in glucagon levels at 120 and 150 min and produced a further decrease in plasma glucose values as compared to the control group injected with insulin. For glucagon values, significant differences were found by the two-way ANOVA for treatment, F(1,66)=47.39, p<0.0001, and time, F(4,66)=10.03, p<0.0001, a significant interaction existing between these two factors, F(4,66)=8.23, p<0.0001; by Duncan's test, significant differences were observed at the 120 and 150 min measurements (Fig. 3A). For glucose values, the two-way
ANOVA showed a significant interaction between time and treatment, F(5,74)=72.16, p<0.0001, and Duncan's test revealed a significant difference at the 90 minute time point (p<0.05) (Fig. 3B).
DISCUSSION
Recently, we synthesized somatostatin analog DPhe-C~'s-Tyr-D-Trp-Lys-Val-C~s-Trp-NH2 (RC-160) which possesses prolonged and enhanced biological activities and shows a high selectivity in inhibiting GH and glucagon release in vivo as compared with its weak suppressive effects on insulin secretion [2, 8, 9]. In normal rats doses of 2 and 10 /zg/kg of this analog completely inhibited the hypoglycemiainduced glucagon secretion [8] and a dose of 10/xg/kg also suppressed the arginine-induced glucagon release (unpublished observations). In the present experiments analog RC-160 inhibited the fasting levels and arginine-elevated levels of glucagon and glucose in diabetic rats. The suppression of glucagon secretion and decrease in plasma glucose levels in diabetic rats may support the view that this analog could be useful as an adjunct to insulin in the treatment of type I diabetes, depending on its propensity to exacerbate the degree of hypoglycemia and to delay recovery from hypoglycemia. In normal man, a recovery from hypoglycemia occurs through a cessation of insulin secretion coupled with the activation of counterregulatory systems [5,14]. On the other hand, in many patients with a long history of type I diabetes the ability to prevent hypoglycemia is impaired because of a blunted or suppressed response to a fall in glucose in addition to the ablation of B cell function [1,14]. In the present experiments our somatostatin analog completely suppressed the hypoglycemia-induced glucagon secretion for a prolonged period. This analog produced an additional reduction in plasma glucose levels during insulin-evoked hypoglycemia, although the decrease caused by the analog was small as compared to the effects of insulin. We could not observe the effect of this analog on the recovery process from insulininduced hypoglycemia in this study, because we used subcutaneous injections of large doses of insulin. Since the duration of the effect of somatostatin analog is shorter than that of lente insulin, the possibility that the analog RC-160 could delay the recovery from insulin-induced hypoglycemia would be small in clinical practice. However, since hypoglycemia is a serious problem in diabetic patients [14], due caution should be exercised not to delay the recovery from the hypoglycemia as well as not to exacerbate it when using somatostatin analogs with potent and prolonged activity together with insulin.
ACKNOWLEDGEMENTS We thank Miss Martha Sampson and Dr. L. Bokser for their valuable experimental assistance. This work was supported by National Institutes of Health Grants DK 07467 and CA 40077 (to A.V.S.) and by the Medical Research Service of the Veterans Administration.
SOMATOSTATIN
ANALOG AND DIABETIC RAT
565
REFERENCES 1. Bolli, G., G. Calabrese, P. De Feo, P. Compagnucci, G. Zega, G. Angeletti, M. G. Cartechini, F. Santeusanio and P. Brunetti. Lack of glucagon response in glucose counterregulation in type I diabetics: absence of recovery after prolonged optimal insulin therapy. Diabetologia 22: 100-105, 1982. 2. Cai, R.-Z., B. Szoke, R. Lu, D. Fu, T. W. Redding and A. V. Schally. Synthesis and biological activity of highly potent octapeptide analogs of somatostatin. Proc Natl Acad Sci USA 83: 1896-1900, 1986. 3. Dimitriadis, G. and J. Gerich. Effect of twice daily subcutaneous administration of a long-acting somatostatin analog on 24hour plasma glucose profiles in patients with insulin-dependent diabetes mellitus. Horm Metab Res 17: 510o511, 1985. 4. Dimitriadis, G., P. Tessari and J. Gerich. Effects of a longacting somatostatin analog on postprandial hyperglycemia in insulin-dependent diabetes mellitus. Metabolism 32: 987-992, 1983. 5. Gerich, J., J. Davis, M. Lorenzi, R. Rizza, N. Bohannon, J. Karam, S. Lewis, R. Kaplan, T. Schultz and P. Cryer. Hormonal mechanisms of recovery from hypoglycemia in man. I. Interaction of glucagon and epinephrin. A m J Physiol 236: E380-E385, 1979. 6. Gerich, J. E., T. A. Schultz, S. B. Lewis and J. H. Karam. Clinical evaluation of somatostatin as a potential adjunct to insulin in the management of diabetes mellitus. Diabetologia 13: 537-544, 1977. 7. Gottesman, I., J. Tobert, R. Vandlen and J. Gerich. Efficacy, pharmacokinetics and tolerability of a somatostatin analogue (L-363,586) in insulin-dependent diabetes mellitus. Life Sci 38: 2211-2219, 1986.
8. Karashima, T., R.-Z. Cai and A. V. SchaUy. Effects of highly potent octapeptide analogs of somatostatin on growth hormone, insulin and glucagon release. Life Sci 41:1011-1019, 1987. 9. Karashima, T. and A. V. Schally. Inhibitory effects of somatostatin analogs on prolactin secretion in rats pretreated with estrogen or haloperidol. Proc Soc Exp Biol Med 185: 6%75, 1987. 10. Lien, E. L., J. Greenwood and D. Sarantakis. Treatment of streptozotocin-diabetic dogs with a long-acting somatostatin analog. Diabetes 28: 491-495, 1979. II. Serrano Rios, M., I. Navascues, J. Saban, A. Ordonez, F. Sevilla and E. Del Pozo. Somatostatin analog SMS 201-995 and insulin needs in insulin-dependent diabetic patients studied by means of an artificial pancreas. J Clin Endocrinol Metab 63: 1071-1079, 1986. 12. Schally, A. V., D. H. Coy, W. A. Murphy, T. W. Redding, A. M. Comaru-Schally, R. Hall, M. D. Rodriguez-Arnao, A. Gomez-Pan, D. Gonzalez-Barcena, R. P. Millar and S. Konturek. Physiological and clinical studies with somatostatin analogs and pro-somatostatin. In: Second International Symposium on Somatostatin, Athens, Greece, edited by S. Raptis, J. Rosenthal and J. E. Gerich. Tubingen: Attempto Verlag, 1984, pp. 188-196. 13. Vranic, M., G. Ross, K. Doi and L. Lickley. The role of glucagon-insulin interactions in control of glucose turnover and its significance in diabetes. Metabolism 25: 1375-1380, 1976. 14. Unger, R. H. The Berson memorial lecture. Insulin-glucagon relationships in the defense against hypoglycemia. Diabetes 32: 575-583, 1983.
0196-9781/88$3.00 + .00
Superactive Somatostatin Analog Decreases Plasma Glucose and Glucagon Levels in Diabetic Rats T S U Y O S H I K A R A S H I M A * A N D A N D R E W V. S C H A L L Y * t I
tEndocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center and *Section of Experimental Medicine, Department of Medicine Tulane University School of Medicine, New Orleans, LA 70146 R e c e i v e d 5 S e p t e m b e r 1987 KARASHIMA, T. AND A. V. SCHALLY. Superactive somatostatin analog decreases plasma glucose and glucagon levels in diabetic rats. PEPTIDES 9(3) 561-565, 1988.--The action of the new analog of somatostatin, DPhe-C~s-Tyr-D-Trp-Lys-Val-C3ls-Trp-NH2 (RC-160), on plasma glucagon and glucose levels was evaluated in streptozotocin-diabetic rats. The effect of this analog on the insulin-induced hypoglycemia in diabetic rats was also investigated in order to evaluate the risk of exacerbating hypoglycemia. Administration of analog RC-160, in a dose of 25 /zg/kg b.wt. SC, inhibited plasma glucagon secretion and decreased plasma glucose levels. This effect also occurred when plasma glucagon and glucose levels were first elevated by arginine infusion, 1000 mg/kg/hr for 30 min. Subcutaneous injection of regular insulin, 15 U/kg b.wt., produced hypoglycemia with a progressive increase in glucagon levels. Analog RC-160 completely suppressed the hypoglycemia-induced glucagon release for up to 150 min after injection of the analog or insulin. A greater decrease in the plasma glucose level was observed in the group treated with insulin and the analog than in the group injected only with insulin. These results indicate that somatostatin analog RC-160 can produce a marked and prolonged inhibition of glucagon release and a decrease in the plasma glucose level in diabetic rats. This analog may be useful as an adjunct to insulin in the treatment of diabetic patients, although caution should be exercised, to prevent hypoglycemia when using somatostatin analogs together with insulin. Somatostatin analog
Diabetes mellitus
Hypoglycemia
T H E effectiveness of glucagon suppression by somatostatin-14 and its analogs has been well documented in diabetic patients [3, 4, 6, 7, 11]. When insulin administration is combined with glucagon suppression, induced by somatostatin or its analog, the dosage of insulin can be reduced [3, 4, 10]. This might be of importance clinically because peripheral hyperinsulinemia, which may contribute to the development of subsequent complications of diabetes mellitus, could thus be avoided [13]. In the treatment of patients with a stable condition of diabetes, it might be worthwhile to try injecting a combination of an intermediate type of insulin and a long-acting somatostatin analog, twice a day, before breakfast and supper. Some somatostatin analogs, which have been developed, could be used for this purpose [7, 10, 12]. We recently synthesized a new somatostatin analog, D-PheC~)s-Tyr-D-Trp-Lys-Val-C}s-Trp-NH2 (RC-160), with prolonged and enhanced activities in addition to a high selectivity in inhibiting glucagon secretion [2, 8, 9]. Since this analog could be useful for treatment of diabetes, we evaluated its effects on the fasting and arginine-elevated glucagon and glucose levels in diabetic rats. Somatostatin analogs could be tried, together with insulin, in patients with a long history of diabetes. Some of these
Glucagon
Blood glucose
patients may experience hypoglycemia due to the dysfunction of their counterregulatory system [1,14]. Since somatostatin analogs can suppress the secretion of glucagon, the main counterregulatory hormone, for a prolonged period, it cannot be excluded that they might produce a serious hypoglycemia in diabetics [13]. We therefore investigated whether the analog would augment the insulin-induced hypoglycemia in diabetic rats. METHOD
Materials Adult male Sprague-Dawley rats weighing 270-350 g were used in all the experiments. Animals were allowed standard diet and tap water ad lib, and were maintained under controlled conditions: 12 hr light/12 hr dark schedule, at 24--2°C. Diabetes was induced by a single IP injection of streptozotocin (Upjohn Research Lab., Kalamazoo, MI), 65 mg/kg b.wt., freshly prepared in 0.05 M citrate and physiological saline, pH 4.5, after an 18 hr fast. Only the rats whose fasting plasma glucose level was in excess of 200 mg/dl were used 4-7 days after the injection of streptozotocin. The number of rats in each group was 8-10.
1Requests for reprints should be addressed to Dr. Andrew V. Schally (151), Veterans Administration Medical Center, 1601 Perdido St., New Orleans, LA 70146.
561
562
KARASHIMA AND S C H A L L Y
o
~e 03 0
(.9
A
400
°°l
80
i
I
I TIME
I (min)
I
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o
n" I,LI N
Z
-
20C
o o
150-
,.J (3
o
100 N
g100-
0
30
60 90 TIME (min)
1 120
50 I 0
I 30
I 60 TIME
I 90
i 120
i 150
180
(min)
FIG. 1. Effect of SC administration of analog RC-160, 25 tzg/kg b.wt., on plasma glucose (A) and glucagon (B) levels in diabetic rats. The levels are expressed as a percentage of the value at the time 0. Absolute basal values of plasma glucose and glucagon were in the range of 226--368 mg/dl and 105-240 ng/ml, respectively. 0: Control group; 0: Group treated with RC-160; *: p<0.05 (by Duncan's test); **: p<0.01 vs. control (by Duncan's test).
160
140
C_12o 0 n." 100
Pent©barbital (Abbot Laboratories, North Chicago, IL), 50 mg/kg b.wt., IP was used as an anesthetic. The anesthesia was maintained with small additional doses of pent©barbital (10 mg/kg b.wt., hourly). One ml of blood sample was drawn from the jugular vein and replaced with the same volume of physiological saline. Arginine solution was infused into the jugular vein using an infusion pump. In the first experiment, the animals were injected with 25 /zg/kg of analog DPhe-C~,s-Tyr-D-Trp-Lys-Val-C#s-Trp-NH2(RC-160) and the samples were collected every 30 min for 2 hr. In the second experiment 25/xg/kg of RC-160 were injected SC and, 30 rain later, arginine HC1 (Sigma, St. Louis, MO) 1000 mg/kg/hr was infused for 30 min. Blood samples were taken at time 0, and 60 and 90 min after the injection of analog. In the third experiment 25/zg/kg of RC-160 and 15 U/kg b.wt. of regular pork insulin (Squibb-Nov©, Princeton, NJ) were given SC and blood samples were taken every 30 min for 150 rain. All samples were collected in chilled tubes containing 1.2 mg EDTA (Sigma) and 500 U of aprotinin (Sigma). Analog RC-160 was synthesized in our laboratory using solid-phase methods and purified by HPLC [2,8]. The peptide was dissolved with physiological saline containing 0.2% bovine
=on 8 0 0 .=J L9
60
40
,
,
0
30
,
,
60 90 T I M E (rain)
,
120
FIG. 2. Effect of SC administration of analog RC-160, 25 /xg/kg b.wt., on plasma glucagon (A) and glucose (B) levels in diabetic rats, elevated by infusion of arginine, 1000mg/kg/hr for 30 min. The levels are expressed as a percentage of the value at time 0. Absolute basal values of plasma glucagon and glucose were in the range of 120-290 ng/ml and 205-480 mg/dl. O: Control; ©: Group treated with RC160; *: p<0.05 vs. control (by Duncan's test); **:p<0.01 vs. control (by Duncan's test).
SOMATOSTATIN ANALOG AND DIABETIC RAT
563
600 LLI
~0 n-
500
LU
N
400
Z
o ¢3 3 0 0 <
,.3 2 0 0
100
I 0
I 30
I 60
i 90
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I, 150
T I M E (rain)
350-
300-
250-
"o
0
r~
120-
100--
8060-
40-
20-
1
I
I
I
I
I
0
30
60
90
120
150
TIME (mln)
FIG. 3. Effect of SC administration of analog RC-160, 25/zg/kg b.wt., on plasma glucagon (A) and glucose (B) levels after SC injection of insulin, 15 U/kg b.wt., in diabetic rats. Plasma glucagon levels are expressed as a percentage of values at time 0. Absolute values of glucagon were in the range of 158--280 ng/ml. Glucose levels are given in absolute values (mg/dl). O: Control; O: Group treated with RC-160; *: p<0.05 (by Duncan's test); **: p<0.01 vs. control (by Duncan's test).
564
KARASHIMA AND SCHALLY
serum albumin. The plasma glucose was determined by a commercial kit using glucose oxidase method (Stanbio, San Antonio, TX). The plasma glucagon was measured by a specific RIA [2,8]. The values of glucose and glucagon, except for glucose levels in the third experiment, were expressed as a percentage of the control [10]. Statistical significance was assessed by Two-Way Analysis of Variance (ANOVA), and by Duncan's multiple range test. RESULTS
As shown in Fig. 1, subcutaneous administration of analog D-Phe-C~'s-Tyr-D-Trp-Lys-Val-C~,s-Trp-NH2 (RC160), at a dose of 25/zg/kg, decreased the basal fasting glucagon level in diabetic rats relative to vehicle-injected controls. By ANOVA, significant differences were found for treatment, F(1,56)=82.1, p<0.0001, and time, F(4,56)=18.39, p<0.0001. The subcutaneous administration of RC-160 also induced a slight but significant reduction in plasma glucose level: two-way ANOVA revealed a significant difference for treatment, F(1,61)=12.92, p<0.001. This difference was largely due to the difference at the 90 min measurement which was significant (o <0.05) by the Duncan's test (Fig. 1). In the second experiment arginine infusion, 1000 mg/kg/hr for 30 min, elevated the fasting plasma glucagon and glucose levels (Fig. 2). A subcutaneous injection of analog RC-160, in a dose of 25/zg/kg, inhibited these increases in glucagon and glucose levels (Fig. 2). Two-way ANOVA for glucagon levels showed significant differences for treatment, F(1,45)=26.93, p<0.0001, and time, F(2,45)=62.94, p<0.0001, and a significant interaction was found between these two factors, F(2,45)=21.34, p<0.0001. By Duncan's test, significant difference was found only at the 60 minute level (p<0.01). For glucose levels, a significant difference was found by ANOVA for treatment, F(1,44)=13.56, p <0.001, and by Duncan's test, a significant difference was detected at the 90 min measurement (p<0.05) (Fig. 2). When the animals were injected SC with 15 U/kg b.wt. of insulin, the plasma glucose was precipitately reduced to hypoglycemia levels, and plasma glucagon levels progressively rose in the third experiment (Fig. 3). Concomitant injection of analog RC-160 completely suppressed the increase in glucagon levels at 120 and 150 min and produced a further decrease in plasma glucose values as compared to the control group injected with insulin. For glucagon values, significant differences were found by the two-way ANOVA for treatment, F(1,66)=47.39, p<0.0001, and time, F(4,66)=10.03, p<0.0001, a significant interaction existing between these two factors, F(4,66)=8.23, p<0.0001; by Duncan's test, significant differences were observed at the 120 and 150 min measurements (Fig. 3A). For glucose values, the two-way
ANOVA showed a significant interaction between time and treatment, F(5,74)=72.16, p<0.0001, and Duncan's test revealed a significant difference at the 90 minute time point (p<0.05) (Fig. 3B).
DISCUSSION
Recently, we synthesized somatostatin analog DPhe-C~'s-Tyr-D-Trp-Lys-Val-C~s-Trp-NH2 (RC-160) which possesses prolonged and enhanced biological activities and shows a high selectivity in inhibiting GH and glucagon release in vivo as compared with its weak suppressive effects on insulin secretion [2, 8, 9]. In normal rats doses of 2 and 10 /zg/kg of this analog completely inhibited the hypoglycemiainduced glucagon secretion [8] and a dose of 10/xg/kg also suppressed the arginine-induced glucagon release (unpublished observations). In the present experiments analog RC-160 inhibited the fasting levels and arginine-elevated levels of glucagon and glucose in diabetic rats. The suppression of glucagon secretion and decrease in plasma glucose levels in diabetic rats may support the view that this analog could be useful as an adjunct to insulin in the treatment of type I diabetes, depending on its propensity to exacerbate the degree of hypoglycemia and to delay recovery from hypoglycemia. In normal man, a recovery from hypoglycemia occurs through a cessation of insulin secretion coupled with the activation of counterregulatory systems [5,14]. On the other hand, in many patients with a long history of type I diabetes the ability to prevent hypoglycemia is impaired because of a blunted or suppressed response to a fall in glucose in addition to the ablation of B cell function [1,14]. In the present experiments our somatostatin analog completely suppressed the hypoglycemia-induced glucagon secretion for a prolonged period. This analog produced an additional reduction in plasma glucose levels during insulin-evoked hypoglycemia, although the decrease caused by the analog was small as compared to the effects of insulin. We could not observe the effect of this analog on the recovery process from insulininduced hypoglycemia in this study, because we used subcutaneous injections of large doses of insulin. Since the duration of the effect of somatostatin analog is shorter than that of lente insulin, the possibility that the analog RC-160 could delay the recovery from insulin-induced hypoglycemia would be small in clinical practice. However, since hypoglycemia is a serious problem in diabetic patients [14], due caution should be exercised not to delay the recovery from the hypoglycemia as well as not to exacerbate it when using somatostatin analogs with potent and prolonged activity together with insulin.
ACKNOWLEDGEMENTS We thank Miss Martha Sampson and Dr. L. Bokser for their valuable experimental assistance. This work was supported by National Institutes of Health Grants DK 07467 and CA 40077 (to A.V.S.) and by the Medical Research Service of the Veterans Administration.
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