Proportional inhibition of glucose-induced insulin release by somatostatin

Proportional

Inhibition

of Glucose-Induced Somatostatin

Gerald J. Taborsky, To

test

the

hypothesis

proportional

inhibitor

release, we examined the release nous

by

output

pancreas basal

from

glucose

the

infusion,

secretion

to the initial rate of

(I = -0.87,

much higher rates of insulin output;

insulin Rapid

of

injection

produced

responses

(AIR).

ment

that

tude

of

a wide

AIR

somatostatin magnitude

of the stimulus.

the inhibitory come

exogenous

endogenous inhibitory stimulation

the

p < 0.005,

of inhibition not

insulin

a decremagnin = 16). by the

stimulus.

presumably

as well, will produce

an

level of glucose

receives.

R

AISING THE PLASMA LEVEL of glucose by glucose infusion not only stimulates insulin release directly but also potentiates the acute insulin release (AIR) elicited by rapid injections of nonglucose stimuli such as isoproterenol,’ arginine,’ secretin, and tolbutamide.4 In our recent study’ examining the effect of somatostatin on this glucose-induced potentiation, we observed that as the magnitude of the AIR to isoproterenol and arginine increased with the plasma glucose level, so did the magnitude of the subsequent inhibition produced by somatostatin. These data suggest that somatostatin is a proportional inhibitor of glucose-potentiated insulin release. This hypothesis implies that exogenous somatostatin, and presumably endogenous somatostatin as well, would exert an inhibitory effect at any level of glucose potentiation that the p-cell receives. Here we test the hypothesis that somatostatin is a proportional inhibitor of glucose-stimulated insulin release. We did this by examining the effect of somatoMetabolism,

Vol.

29,

No.

12 (December),

1980

METHODS

Insulin

Sixteen adult male dogs of mixed breed were pretreated with morphine sulfate (30 mg s.c.) and then anesthetized with sodium pentobarbital (30 mg/kg i.v.). Rectal temperablood gases were held within the normal range by adjusting the rate and tidal volume of the respirator. A midline laparotomy was performed and the blood flow of the superior pancreaticoduodenal vein (SPDV) was shunted past a sampling port, through an electromagnetic flowmeter, and then to the portal vein, as previously described.b Samples of superior pancreaticoduodenal vein plasma were drawn from this flow circuit for determinations of insulin concentration. Coupling this measurement with blood flow allowed calculation of insulin output from the right lobe of the in situ pancreas (see below). A cannula was also introduced into the femoral vein for the infusion of dihydrosomatostatin (courtesy of Dr. Jean Rivier, Salk Institute) and glucose when appropriate. A second cannula was introduced into the femoral artery to allow measurement of arterial plasma glucose. A l-hr stabilization period followed surgery before baseline samples were drawn. Two types of experiments were performed. The first series of experiments were designed to evaluate the inhibitory effect of somatostatin (SRIF) (I .7 pg/min x 30 min) over a wide range of basal insulin outputs. The second series of experiments was designed to evaluate the inhibitory effect of this dose of somatostatin over higher range of insulin outputs stimulated by various rates of glucose infusion (I --6 mg/kg/min). The second experimental series was done on the same day and in the same dogs as those used for the first

cannot be overand

AND

of Basal and Stimulated

tures were regulated at 38.5 k 0.5OC by a thermal blanket;

with

of glucose

effect at any physiologic that the &cell

by

These data suggest that size

MATERIALS

Assessment Output

of

produced

decreases,

somatostatin,

somatostatin

acute

to the original

effect of somatostatin

by increasing

Thus,

of

statin on the insulin release stimulated by the endogenous glucose level as well as a wide range of higher glucose levels achieved by glucose infusion or injection.

n = 16).

rate

2 or 20 g of

produced

(r = -0.70.

increases,

initial

p < 0.01,

range

again,

produced

the

Somatostatin

Thus, the absolute amount

mg/kg/min

of either

was proportional

the

l-8

output

with

(r = -0.68.

intravenous

glucose

insulin

correlated

secretion

of

p < 0.001,

produced

decrement

infusions

by

Phillip H. Smith

(1.7 canine

Glucose

somatostatin

by

and

of basal

of the

n = 18). the

on

or

infusions

lobe

a

by endoge-

a decrement

right

that was proportional

insulin

either

Somatostatin

x 30 min) produced

insulin

is

insulin

the effect of somatostatin

(basal),

injection.

pglmin

somatostatin

glucose-induced

of insulin stimulated

signals

glucose

that

of

Jr.

Insulin Release

From the Department of Medicine, and the University of Washington, School of Medicine, and the Division of Endocrinology and Metabolism. Veterans Administration Medical Center, Seattle. Wash. Receivedfor publication March 27, 1980. Supported in part by the Veterans Administrarion, the Diabetes Research Center (AM 170471. and USPHS grants AM 05409, AM 12829. AM 20284. AM 25325. Address reprint requests to Dr. Gerald J. Taborsky. Jr., Division of Endocrinology and Metabolism (I 51). V-4 Medical Center. 4435 Beacon Avenue South, Seattle, Wash. 98108. 0 1980 by Grune & Stratton, Inc. 0026.-0495/80/29/ 2ZO009$01.00/0

1253

1254

experimental series. Glucose was infused into the femoral vein 90 min before the second somatostatin infusion to allow insulin output to stabilize. The glucose infusion was continued during the infusion of somatostatin. Blood samples were drawn at 30, 15,and 0 minutes before; at 5, 10, 20 and 30 min during; and then at 5, 10, 15, and 30 min after the infusion of somatostatin. The hematocrit was determined, and then the plasma was separated and frozen for later analysis. Plasma insulin values were measured by radioimmunoassay using a modification of the double antibody technique of Morgan and Lazarow.’ Glucose was estimated by the ferricyanide method using the Technicon AutoAnalyzer. insulin (IRI) output from the right lobe of the canine pancreas was calculated according to the formula: IRI output (rU/min) = IRI concentration in the SPDV plasma (&/ml) x (1-hematocrit) x blood flow in the SPDV (ml/min). The decrement of insulin output produced by the infusion of somatostatin was calculated by subtracting the mean of the output values at IO, 20, and 30 min during the infusion from the mean of the baseline values 30, 15, and 0 min before the infusion. The statistical significance of the inhibition was evaluated using Student’s paired t test. Standard linear regression analysis was used to correlate the decrement produced by somatostatin with the preceding insulin output. The slopes of the regression lines were compared using the F test.

Assessment of Acute Insulin Release A third group of eight fasted dogs was anesthetized with sodium pentobarbital as before. Catheters were inserted into a femoral artery for blood sampling and into a femoral vein for the infusion of somatostatin and/or injection of glucose. These experiments examined the inhibitory effect of the same dose of SRI F (I .7 rg/min) over a wide range of acute insulin responses stimulated by the rapid injection of either 2 or 20 g of glucose. On the day of the control study, blood samples were drawn 15, 10, 5, and 0 min before and at 2, 3, 4, 5, 7, IO, 15, and 30 min after the rapid injection (less than 30 set) of 2 g of glucose. Only the value immediately preceeding the injection of glucose was used as the baseline for the calculation of the acute insulin response (see below). One hour after the first injection, 20 g of glucose were injected. Blood samples were drawn as before. On the day of the experimental study (approximately 2 wk later) the 2 and 20 g glucose pulses were repeated in the same dogs during infusion of SRIF at 1.7 rg/min. Somatostatin was infused via the femoral vein for 15 min starting 5 min before each glucose pulse. The acute insulin response (AIR) to the injected glucose was assessed by calculating the area under the arterial insulin time-curve for the 10 min following injection above the baseline insulin level. This baseline level was taken 5 min after the start of the somatostatin or saline infusion, immediately preceeding the injection of glucose. The timing of the baseline sample was designed to eliminate any inhibitory effect of somatostatin on the AIR that may be due solely to an effect on basal, rather than stimulated, insulin release. The statistical significance of changes in this incremental area was evaluated using Student’s paired t test.

TABORSKY

AN0

SMITH

Fig. 1. Effect of a constant dose of somatostatin (SRIF, 1.7 Mg/min i.v.1 on basal insulin output from the right lobe of the canine pancreas in three individual experiments.

RESULTS

Basal and Stimulated Insulin Output In the 16 dogs studied, basal insulin output from the right lobe of the pancreas ranged from 966 to 12,313 pU/min as a linear function of fasting plasma glucose (r = +0.608, p < 0.01, n = 16). The intravenous infusion of somatostatin (1.7 pg/min x 30 min i.v.) produced an inhibition of insulin output that appeared to increase with the basal output level as illustrated by the data from three of these experiments (see Fig. 1). This impression was confirmed by analyzing the data from all 16 experiments: the decrements of insulin output produced by infusion of somatostatin correlated linearly with the basal rate of hormone output (r = -0.87, p -C0.001, n = 16; Fig. 2). Glucose infusions of 1-6 mg/kg/min proO-

- 2000; 9 ‘s a

-4ooo-

$

-6OOO-

q -6OOO-

y= -0.58x-303 n= 16

p(.OOl -10.000-

I 0

I I0.000

I 5000 Basal

IRI

Oulput,

( 15,000

pU/min

Fig. 2. Relation between basal insulin output and the mean decrement of that output produced by the intravenous infusion of somatostatin (SRIF. 1.7 rzg/min i.v.1 in 16 dogs. The data from the experiments shown in Fig. 1 are circled.

MPAIRED INSULIN SECRETION

1255

Glucose. 20 am.

60

Ji

PC 01

-36.000

0

0

24,000 48.000 Stimuhfed [RI ou,put, p"/mln

1 72,000

Relation between stimulated insulin output and Fig. 3. the absolute decrement of that output produced by an intravenous infusion of SRIF (1.7 pg/min x 30 min). Glucose was infused at l-3 (A) and 6 (0) mglkglmin to produce a steady-state elevation of insulin output.

i l$ 20 z -

/:,I, -15

duced insulin outputs with a five-fold greater range: 4600 to 67.155 pU/min. Plasma glucose was increased from a baseline of 120 f 4 mg/dl (X + SEM) before glucose infusion to a level of 2 17 i- 15 mg/dl after 1 hr of glucose infusion at 6 mg/kg/min. The addition of somatostatin after 90 min of glucose infusion produced decrements of insulin output from the pancreas that again correlated linearly with the preceding insulin output r = -0.68, p < 0.01; Fig. 3). The slope of this regression line (-0.43) was not significantly different from the slope of the line (-0.58) relating basal insulin output to the decrement produced by somatostatin (Fig. 2). Infusions of somatostatin at 1.7 pg/min produced no significant change of blood flow in the superior pancreaticoduodenal vein. Acute Insulin Release On the day of the control study, when the dogs did not receive SRIF, the rapid injection of glucose (2 g i.v.) increased arterial plasma insulin from a basal level of 7 * 1 pU/ml (X + SEM) to 19 & 1 pU/ml by 2 min after the injection (Fig. 4). Plasma glucose increased from a basal level of 125 k 1 I mg/dl to 174 _+9 mg/dl over the same time period. The incremental area under the insulin curve for the 10 min following glucose injection was 105 + 16 pU/ml x min. This area was used as an index of the acute insulin response to glucose (see Materials and Methods). On the day of the experimental study, the infusion of SRIF (1.7 pg/min x 15 min) decreased basal arterial insulin from 10 + 2 pU/ml to 6 t I pU/ml within 5 min and

0

15

30

-15

, 0

15

30

MINUTES Fig. 4. Time course of mean arterial insulin responses (2 i SEM. n = 81 to rapid intravenous injections of either 2 g of glucose (left panel) or 20 g of glucose (right panel) in the absence (0) or presence of (0) of SRIF (1.7 fig/min x 15 min).

decreased the increment of insulin area following the 2 g glucose pulse (area during SRIF = 29 _t 11 pU/ml x min; p < 0.00 1). During control studies, the rapid injection of 20 g of glucose (i.v.) increased arterial plasma insulin from 14 k 1 pU/ml to 51 -t 5 pU/ml by 2 min after the injection (see Fig. 4). Plasma glucose increased from a prestimulus level of 145 * 29 mg/dl to 639 + 29 mg/dl over the same time period. The increment of insulin area following the 20 g glucose pulse was 31 1 * 34 $J/ml x min. During experimental studies, the infusion of SRIF (1.7 pg/min x 15 min) decreased the prestimulus level of arterial insulin from 16 L 3 wU/ml to 9 + 1 pU/ml within 5 min (p < 0.005) and decreased the increment of insulin area following a 20-g glucose pulse (area during SRIF = 127 t pU/min; p c 0.005). The incremental areas produced by these acute glucose stimuli ranged from 9 to 414 pU/ml x min. The decrement of these insulin areas produced by the infusion of SRIF correlated with the magnitude of the area before SRIF (r = -0.702; p < 0.005; see Fig. 5). The slope of this regression line (-0.52) was not significantly different from the slope of the lines relating either basal (-0.58) (Fig. 2) or stimulated (-0.43) (Fig. 3) insulin output to the respective decrements produced by SRI F.

1256

TABORSKY AND SMITH

r=-0.702 y=-0.52 ll’16 P’ 0.005

a -300

’ 0

II)

I loo

-21

I 200 ACUTE IRI (pU/ml

AREA X min

I 300 O-IO’

I 400

Y

1

Fig. 5. Relation between the incremental area under the insulin curve (see Fig. 4) for the 10 min following the rapid injection of either 2 g (ml or 20 g (0) of glucose and the decrements of that area produced by intravenous infusion of SRIF (1.7 Ng/min x 15 min).

DISCUSSION

These studies were designed to test the hypothesis that somatostatin is a proportional inhibitor of glucose-induced insulin release. If this hypothesis is true, the absolute amount of inhibition produced by somatostatin should increase with the stimulation for insulin release. This pattern accurately describes the observed effect of somatostatin on basal insulin output: the absolute decrement of insulin output produced by somatostatin was proportional to the initial rate of basal insulin output. Since it was possible that the range of endogenous stimulation was insufficient to adequately test this hypothesis, exogenous glucose was infused to markedly increase the range of insulin output. Somatostatin continued to produce an inhibitory effect that increased with the stimulation for insulin release. We also assessed the inhibitory effect of somatostatin on the acute insulin release stimulated by injections of glucose with which we were able to transiently achieve higher levels of plasma glucose than those produced by glucose infusion. Somatostatin again produced an inhibitory effect that increased with the stimulation of acute insulin release. Furthermore, the slopes of the three regression lines relating the decrements produced by somatostatin to the original magnitude of insulin release were not significantly different whether the secretion of insulin was stimulated by endogenous signals (basal) or stimulated by glucose infusion or injection. Thus, somatostatin appeared to inhibit the same proportion of insulin release indepen-

dent of the’type, as well as the magnitude, of glucose stimulation. We conclude that, over the range of glucose stimulation studied, somatostatin is a proportional inhibitor of glucose-induced insulin release. Somatostatin appears to be a proportional inhibitor of the insulin release potentiated by glucose as well as that directly stimulated by glucose. This hypothesis is supported by data from our previous study’ in which it was shown that glucose infusions increased the magnitude of the acute insulin release (AIR) to a constant dose of either isoproterenol or arginine. We also found that the greater the AIR, due to the potentiating effects of glucose, the larger the decrement of the AIR produced by a constant dose of somatostatin. Those data coupled with these presented here suggest that somatostatin may achieve its proportional inhibition of insulin release by simply interferring with the intracellular signals generated by glucose at the P-cell that are capable of mediating direct stimulation of insulin release as well as potentiating acute insulin release by nonglucose signals. It is likely that the inhibitory action of somatostatin on other endocrine cells is also mediated by interference with a common intracellular signal that is necessary for peptide hormone release. Other investigators have examined the effect of somatostatin on the dose-response relationship between glucose and insulin release. Fujimoto* measured insulin secretion from cultured rat islets during incubation with 5.5 and 16.5 mM glucose in the presence and absence of somatostatin. He reported that the decrement produced by equivalent doses of somatostatin was larger at 16.5 mM glucose than at 5.5 mM glucose. These in vitro data thus complement the in vivo data reported here. Basabe et al.,9 using slices of rat pancreas, reported that the decrease of insulin secretion produced by somatostatin either remained constant or increased as glucose was added to the incubation media. They concluded that somatostatin is a noncompetitive inhibitor of glucose-induced insulin release because increasing glucose stimulation did not overcome the inhibitory effect of somatostatin. In contrast, Efendic, Luft, and Clara” suggested that somatostatin might be a competitive inhibitor since they observed little inhibition at a high rate of primed glucose infusion. However, they also

IMPAIRED

INSULIN

SECRETION

1257

observed little inhibition at the lowest rate of primed glucose infusion where a competitive inhibitor should have had maximal effect. Thus, the pattern of inhibition they observed is not easily classified as either competitive or noncompetitive. The distinction between competitive and noncompetitive inhibition of insulin release is difficult to make in this animal model because it is best observed at maximal stimulation of insulin release. Such stimulation is not easily achieved by glucose infusion in vivo since prolonged hyperglycemia increases insulin biosynthesis” and, thus, the amount of insulin available for release. In addition, the renal threshold for glucose and the action of the released insulin to increase the intracellular glucose transport limits the amount of hyperglycemia one can achieve. Stimulation of acute insulin release by rapid glucose injection circumvents some of the problems; however, the complexity of stimulussecretion coupling still casts doubt on the use of the biochemical designations, competitive and

non-competitive, for classifying inhibition of insulin secretion. Thus, the present data do not allow us to definitively classify somatostatin as either a competitive or noncompetitive inhibitor of insulin release. These data do, however, place an important constraint on proposed mechanisms for somatostatin action: they should simulate an inhibition whose absolute magnitude increases over the physiologic range of antecedant glucose stimulation. This hypothesis implies that glucose stimulation will magnify rather than overcome the inhibitory effects of somatostatin.

ACKNOWLEDGMENT The authors thank Dr. Daniel Porte, Jr. for valuable discussions, and Dr. Jean Rivier of the Salk Institute for his generous gift of the dihydrosomatostatin used in these studies; Robert Guest and Scott Horton for their technical and experimental assistance; Howard Beiter, Barbara O’Neill, Karen Guest, Connie Holmes, and Rogene Bennett for the measurements of glucose and insulin; Pat Jenkins for secretarial assistance.

REFERENCES 1. Halter JB, Graf RJ, Porte D Jr: Potentiation of insulin secretory responses by plasma glucose levels in man: Evidence that hyperglycemia in diabetes compensates for impaired glucose potentiation. J Clin Endocrinol Metab 48:946-954. 1979 2. Palmer JP. Walter RM, Ensinck JW: Arginine-stimulated acute phase of insulin and glucagon secretion. I. In normal man. Diabetes 24:735-740, 1975 3. Lerner RL, Porte D Jr: Studies of secretin-stimulated insulin responses in man. J Clin Invest Sl:2205-2210, 1972 4. Efendic S, Luft R, Cerasi E: Quantitative determination of the interaction between epinephrine and various insulin releasers in man. Diabetes 27:319-326, 1978 5. Taborsky GJ Jr, Smith PH, Halter JB, et al: Glucose infusion potentiates the acute insulin response to nonglucose stimuli during the infusion of somatostatin. Endocrinology 105:1215-1220, 1979 6. Taborsky GJ Jr, Smith PH. Porte D Jr: Differential effects of somatostatin analogues on cy- and &cells of the pancreas. Am J Physiol236:El23-El28, 1979

7. Morgan CR, Lazarow A: Immunoassay of insulin: Two antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12: I 15-l 26, 1963 8. Fujimoto WY: Somatostatin inhibition of glucose-. tolbutamide-, theophylline-, cytochalasin B-. and calciumstimulated insulin release in monolayer cultures of rat endocrine pancreas. Endocrinology 97: 1494-l 500, 1975 9. Basabe JC, Crest0 JC, mode of action of somatostatin nology lOl:l436-1443, 1977

Aparicio N: Studies on the on insulin secretion. Endocri-

IO. Efendic S, Luft R, Clara A: Studies on the mechanism of somatostatin action on insulin release in man. II. Comparison of the effects of somatostatin on insulin release induced by glucose, glucagon and tolbutamide. Acta Endocrinol 8 I :743-752. 1976

I I. Tanese T, Lazarus NR, Devrim S, et al: Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans. J Clin Invest 49: I394~1404, 1970