GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
31,249-256
(1977)
Glucose and Amino Acid-Stimulated Insulin Release in Vivo in the European Silver Eel (Anguilla anguilla L.) BERNARD W. INCE AND ALAN THORPE Department
of Zoology & Comparative Physiology, Queen (University of London). Mile End Road, London, El 4NS, United Kingdom
Mary
College
Accepted October 22, 1976 Glucose and amino acid-stimulated insulin release was studied in vivo in cannulated European silver eels (Anguilla anguilla). Glucose-stimulated insulin release was dose dependent over a range of glucose loads (lo-100 mg/kg) while higher doses (300 and 500 mg/kg) produced no greater increments than 100 mg/kg. Arginine and lysine injections of 10, 25 and 100 mg/kg caused greater significant increases in plasma insulin levels than glucose at the same dose levels. Histidine (10 and 25 mglkg) caused a small but significant reduction in the plasma insulin level. Simultaneous injection of arginine (100 mglkg) and glucose (100 mgtkg) caused an increase in plasma insulin which was sustained for 60 min and remained significantly above the controls over the 360 min sampling period. Total insulin secretion appeared to be significantly greater over the entire sampling period than when arginine and glucose were injected alone.
In mammals and man, the factors which stimulate, modulate or inhibit insulin secretion have been studied in great detail both in viva and in vitro (cf. Gerich et al., 1976). In fishes, the few reports deal mainly with nutritional factors affecting insulin secretion. Thus, using a morphological approach it has been shown that glucose loading in certain fish species causes degranulation of the B-cells, implying glucose-stimulated insulin release (Khanna and Mehrotra, 1969; Khanna and Rekhari, 1972; Khanna and Gill, 1973). More quantitative data have been obtained by the use of radioimmunoassay of endogenous insulin levels. Tashima and Cahill (1968) observed that the serum insulin level of protein-fed toadfish, Opsanus tau, was significantly higher than in starved fish while oral glucose administration gave no response. Similar opposing effects of feeding and starvation on insulin levels have been observed more recently in goldfish, Carassius auratus (Patent and FOB, 1971), and in cod, Gadus morhua, and
rainbow trout, Salmo gairdneri (Thorpe and Ince, 1976). Patent and FOB (1971) reported in addition, that glucose and leucine, but not arginine, stimulate insulin release from the islets of toadfish in vitro and that the leucine effect is enhanced by the simultaneous addition of glucose. In the present investigation, the effects of glucose, arginine, lysine and histidine on plasma immunoreactive insulin levels have been studied in vivo in cannulated European silver eels, Anguilfa anguilla. MATERIALS
Maintenance Technique
AND METHODS
of Fish and Cannulation
European silver eels (A. anguilla) were purchased from a local dealer and maintained in polyethylene tanks supplied with a constant flow of fresh, aerated water. The mean weight of the eels used was 432 g (range 300-510 g). The experiments were performed between the period February to May 1976 when the tank water temperatures ranged from 9 to 13°C. Eels used in the present study had been fasting for 2-3 months prior to purchase and remained in this state
249 Copyright All rights
@ 1917 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
CtOl6-6480
250
INCE
AND
for the duration of the experiments. Prior histological investigation in such specimens has shown no evidence for degeneration of the endocrine pancreas (Ince and Thorpe, 1974). After a 2 day acclimation period, the fish were cannulated as described previously (Ince and Thorpe, 1974) and left for a further 3 days to allow for recovery from the stress effects caused by the operation (Thorpe and Ince, 1976). There were no significant variations in plasma glucose, amino acid nitrogen or insulin as related either to body weight or the duration of the period of this study.
Analytical
THORPE
levels of 10, 25 and 100 mgikg body weight and L-histidine at dose levels of 10 and 25 mg/kg body weight. It was not possible to use higher dose levels of histidine since these produced immediate and obvious signs of distress.
Statistical
Analysis
Paired t tests were used to test the significance between the means of the two preinjection control values and each of the values obtained following glucose or amino acid administration. The differences were considered significant when P < 0.05.
RESULTS
Techniques
Effects of Serial Blood Sampling on Plasma Glucose. Amino Acid Nitrogen and Insulin Levels Figure 1 shows that serial blood sampling does not induce any significant fluctuations in the levels of plasma glucose, amino acid nitrogen or insulin over a 360 min period. The control plasma insulin levels obtained Glucose and Amino Acid Administration in the present study, however, ranged from and Blood Sampling 2.40 to 3.80 rig/ml which is approximately Glucose and amino acids were dissolved in 2-3 times greater than those reported earphosphate-buffered. heparinised saline (pH 7) and lier from a study carried out between Ocadministered as single, rapid intra-arterial injections tober 1975 and January 1976; 1.12 + 0.10 through the cannula at varying dose levels. Blood rig/ml (11 animals) (Thorpe and Ince, 19761. samples (0.45 ml) were removed after 15. 30, 45, 60, Similarly, control plasma glucose levels 120, 180 and 360 min and a corresponding volume of were approximately 20-30 mg/dl greater buffer was replaced. Control levels of plasma glucose, amino acid nitrogen and insulin were assayed in than those previously reported, 42.2 t 5.0 two blood samples taken at 60 and 0 min prior to the mg/dl ( 11). It is not easy to ascribe precise injection of glucose or amino acids. D-Glucose was administered at dose levels of 10. reasons for these differences since assay techniques, blood sampling method and 25, 50, 100, 300 and 500 mglkg body weight. conditions were identical in L-Arginine and L-lysine were administered at dose experimental Plasma glucose, amino acid nitrogen and insulin were assayed in duplicate from a single plasma sample taken at each time interval from individual fish. The assays were performed either immediately or within 2 days from samples stored at -20°C. Details of the assay methods used have been given in earlier publications (Ince and Thorpe, 1974; Thorpe and Ince, 1976).
16) t
$I $GJ
1 k-H--: 0l5304560
(6)
t 120 time (minutes)
1 I 180
-_i 360
FIG. I. Effects of serial blood sampling on plasma glucose, amino acid nitrogen and insulin levels. Mean ‘t SE; number of animals in parentheses. The results show that serial blood sampling in cannulated silver eels over 360 min does not alter significantly the levels of plasma glucose, amino acid nitrogen and insulin.
INSULIN
RELEASE
IN
2.51
EELS
silver eels obtained from various localities around the British Isles.
the two studies. It may be that seasonal influences have a bearing on plasma glucase and insulin levels even in a naturally fasting animal such as the silver eel, but the differences could equally be of an intraspecific nature (Mazeaud, 1973), since we are dependent upon a commercial supply of
Effects of Intra-arterial on Plasma Glucose
Glucose Loading and Insulin Levels
Figures 2a and 2b show the response curves for plasma glucose and insulin given
11 d
1 -60
0
1.5
30
45
120
60 minutes
l&O
360
port-injection
FIG. 2a. Effects of intra-arterial glucose loading on plasma glucose and insulin levels. Mean ? SE; number of animals in parentheses; (0, A, W) glucose loads of IO, 25 and 50 mg/kg, respectively. The relationship between glucose levels and plasma insulin response is evident. Maximal insulin levels occur 30 min after injection and are significantly different from the controls. 900 ,700. e P -500. 4 s yoo 100. Oi
I
-60
0
15 30 45 60
120 minutes
180
360
port-injection
FIG. 2b. Effects of intra-arterial glucose loading on plasma glucose and insulin levels. Mean _CSE; number of animals in parentheses; (0, & 0) glucose loads of 100, 300 and 500 mgikg respectively. A significant elevation of plasma insulin occurring after 30 min is seen at 100 mg/kg, while at higher glucose doses, the elevation of insulin above control levels, although no greater than at 100 mg/kg, occurs after only I5 min. Recovery of plasma insulin to near-control levels occurs after 360 min at all doses despite the significantly elevated plasma glucose levels at this time
2.52
INCE
AND
by lo,25 and 50 mg/kg and 100,300 and 500 mg/kg glucose loads, respectively. At 10, 25, 50 and 100 mg/kg the mean maximal increases in plasma insulin above control levels after 30 min were 0.90, 1.51, 2.17, and 3.40 rig/ml, respectively. For glucose loads of 300 and 500 mglkg the mean maximal increases were 3.70 and 3.00 rig/ml, respectively, after only 15 min. These increased plasma insulin concentrations were significant at all dose levels but a log-linear relationship between dose of glucose and peak insulin values was observed only up to 50 mg/kg. At all dose levels of glucose, insulin returned to control values within the 360 min period despite the fact that glucose levels remained elevated at loads greater than 25 mg/kg.
THORPE
hyperglycaemia of 12 mgidl above control values but was without effect on plasma glucose at dose levels of 10 and 25 mg/kg. Effects of Intra-arterial Histidine Loading on Plasma Glucose, Amino Acid Nitrogen and Insulin Levels Histidine injections of 10 and 25 mg/kg caused a small but significant mean reduction in plasma insulin levels of between 0.70 and 0.93 rig/ml (Fig. 5). The response was slow, reaching a nadir between 60 and 120 min after injection with gradual recovery to control levels thereafter. No significant alterations in plasma glucose concentrations were observed at either dose level and only a small increase in amino acid nitrogen was seen after 15 min at 2.5 mg/kg.
Effects of Intra-arterial Arginine Loading Effects of Simultaneous Intra-arterial on Plasma Glucose, Amino Acid Arginine and Glucose Loading on Nitrogen and Insulin Levels Plasma Glucose, Amino Acid Nitrogen and Insulin Levels Arginine injections of 10, 25 and 100 Simultaneous administration of arginine mg/kg caused significant mean increases in plasma insulin above control levels of 1.67, ( 100 mg/kg) and glucose ( 100 mg/kg) caused 3.12 and 6.25 ngiml, respectively, after 15 a significant and sustained elevation of min (Fig. 3). Subsequent recovery of plasma insulin of 12.00 &ml above control plasma insulin to control levels was pre- levels between 15 and 60 min after injection ceded by that of amino acid nitrogen at 25 (Fig. 6). The mean sum of increments over and 100 mg/kg. A significant although tran- the entire sampling period was significantly sient hyperglycaemia of 10 mg/dl above greater than the summed insulin response control values occurred with arginine at 100 given by arginine or glucose alone over the mg/kg, while no significant effects on same period. Between 60 and 360 min after injection, plasma insulin levels fell slowly plasma glucose were observed at the lower dose levels. by a total of only 3.55 rig/ml so that after 360 min the plasma insulin levels were still Effects of Intra-arterial Lysine Loading on significantly greater than the controls. The Plasma Glucose, Amino Acid Nitrogen simultaneous injection of arginine and gluand Insulin Levels cose was followed by a significantly accelerated clearance of the excess glucose Lysine injections of IO,25 and 100 mg/kg when compared with the clearance of glucaused significant mean increases in plasma insulin above control levels of 2.43, 5.35 cose from plasma following a single glucose and 10.86 rig/ml, respectively, after 15 min load of 100 mg/kg. The plasma clearance of amino acid nitrogen, however, was not sig(Fig. 4) with the plasma insulin clearance closely resembling that for amino acid ni- nificantly altered by a simultaneous injectrogen at all dose levels. Lysine at 100 tion of arginine and glucose when compared with arginine alone. mg/kg caused a significant but transient
INSULIN
RELEASE
IN
253
EELS
360 minutes
post-
injection
FIG. 3. Effects of intraarterial arginine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean f SE; number of animals in parentheses; (0, A, I) Arginine loads of 10, 25 and 100 &kg, respectively. Arginine at all doses causes significant maximal elevation of plasma insulin after 15 min. At 100 mg/kg, however, a reduced rate of plasma insulin clearance is seen when compared with that of amino acid nitrogen. A significant but transient hyperglycaemia occurs with an arginine load of 100 mg/kg.
DISCUSSION pling frequency used in the present study. The present data show that glucose, ar- Thus, the increased rate of fall of plasma ginine and lysine stimulate insulin release in glucose which follows the higher loads (Fig. viva in silver eels and that the initial rise in 2b) cannot be due to insulin alone. As has been shown previously (Ince and Thorpe, plasma insulin levels following the administration of the amino acids is greater than for 1974), the terminal portion of the glucose loading curve (assimilation phase) shows glucose at the same dose level. only a slow recovery to initial levels and The glucose loading data indicate a direct relationship between increasing glucose from the present data it can be seen that load and the rate of fall of plasma glucose during this time insulin levels are falling during the initial 30 min period. At this correspondingly. Data from mammalian studies show that time, over the range lo-100 mg/kg, there is a corresponding increase in the concen- certain amino acids stimulate not only the tration of plasma insulin which could ac- release of insulin but also growth hormone count for the accelerated removal of the and glucagon (cf. Gerich er al., 1976). Since excess glucose from the blood. The level of the latter hormones are also known to plasma insulin attained after a glucose load stimulate insulin secretion, the effect of inof 100 mg/kg was not seen to be signifi- jected arginine and lysine in the present study (Figs. 3 and 4) may be due to a comcantly exceeded if the dose was increased to 300 or 500 mg/kg, at least with the sam- bination of secondary hormonal influences
254
INCE
minutes
AND
THORPE
post-injectbn
FIG. 4. Effects of intra-arterial lysine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean f SE; number of animals in parentheses; symbols and dose levels as in Fig. 3. Lysine at all doses causes a significant maximal elevation of plasma insulin after 15 min. A significant but transient hyperglycaemia occurs with lysine at 100 mg/kg.
FIG. 5. Effects of intra-arterial histidine loading on plasma insulin levels. Mean + SE; number of animaIs in parentheses; (0, A) histidine loads of 10 and 25 mgikg, respectively. Histidine at both doses causes a significant but transient reduction in plasma insulin.
INSULIN
RELEASE
IN
255
EELS
1
-60
0
15 30 45 60 minutes
120 port-injeciion
180
360
FIG. 6. Effects of simultaneous intraarterial glucose and arginine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean 2 SE; number of animals in parentheses; (0) simultaneous injection of arginine (100 mg/kg) and glucose (100 mg/kg). The effects of glucose (0) and arginine alone (m), shown earlier, are given here for comparison. Simultaneous injection of arginine and glucose results in a significant elevation of plasma insulin over the entire sampling period. The total insulin secretion appears significantly greater than when either is injected alone. Plasma clearance of amino acid nitrogen remains unchanged but excess glucose removal is accelerated.
and a direct interaction of the amino acids with the B-cells. In addition, the arginine and lysine-induced hyperglycaemia, seen at 100 mglkg, results presumably from the influence of released glucagon which is known to be glycogenolytic in eels (Larsson and Lewander, 1972; Ince, 1975). By comparison with the present data, Patent and FoA (1971) reported that both glucose and leucine, but not arginine, caused significant insulin release from the islets of toadfish, 0. tau in vitro, whereas oral glucose loading in the same species failed to elicit insulin release (Tashima and Cahill, 1968). Patent and FOB (1971) also reported a synergistic effect of glucose and leucine on insulin release from toadfish islets in vitro. Likewise in the present study, the simul-
taneous injection of arginine and glucose appeared to cause a significantly greater total insulin secretion over the entire sampling period than when either was injected alone (Fig. 6). However, this apparent synergism between glucose and arginine in vivo may have been caused by concomitant release of glucagon (or other hormones) stimulating the B-cells as well as by the direct effects of glucose and arginine. It is interesting to note that in silver eels, histidine does not stimulate insulin release but in fact causes a reduction in plasma insulin levels (Fig. 5). Similarly, in man, histidine has been shown to be the least potent of the amino acids in stimulating insulin release (Fajans and Floyd, 1972). The reasons for the marked difference in its ac-
256
INCE AND THORPE
tions when compared with other amino acids such as arginine and lysine, are unknown. The plasma clearance of arginine and lysine as judged by total plasma amino acid nitrogen is very similar at all dose levels (Figs. 3 and 4). It is of interest, therefore, that the shape of the plasma insulin curves following their injection differs markedly. It is likely that the sustained elevation of plasma insulin following arginine injection, either alone or with glucose, is due to a greater total output of the hormone caused by a different response of the B-cells to that following lysine injection. Alternatively, it could be argued that the differences between the curves are due either to insulin distribution and degradation phenomena as has been emphasised by Yalow and Berson (1965) or to secondary hormonal influences on the B-cell arising from the initial amino acid stimulus. The latter idea is supported by the observation that hypoglycaemia during pronounced and prolonged hyperinsulinaemia is absent, suggesting that glucagon or other insulin antagonists may be operating to maintain plasma glucose at a normal level. ACKNOWLEDGMENT This investigation forms part of a research project supported by a grant to one of us (A. T.) from the Science Research Council, England, to whom our thanks are due.
REFERENCES Fajans, S. S., and Floyd, J. C., Jr. (1972). Stimulation of islet cell secretion by nutrients and by gastrointestinal hormones released during digestion.
In “Handbook of Physiology” (D. F. Steiner and N. Freinkel, eds.). Vol. 1, Sect. 7. pp. 473-493. Amer. Physiol. Sot., Washington, D.C. Gerich, J. E., Charles, M. A., and Grodsky, G. M. (1976). Regulation of pancreatic insulin and glucagon secretion. Ann. Rev. Physiol. 38, 353388. Ince, B. W. (1975). Studies on the hormonal control of metabolism in teleosts. PhD thesis, University of London, England. Ince, B. W.. and Thorpe, A. (1974). Effects of insulin and of metabolite loading on blood metabolites in the European silver eel (Anguillu ungttilh L.). Gen. Comp. Endocrinol. 23, 460-471. Khanna, S. S., and Gill. T. S. (1973). Effect of glucose loading on the blood glucose level and histology of the principal islets in Channa puncfatus. Endocrinology (Japan) 20, 375-383. Khanna, S. S., and Mehrotra. B. K. (1969). Effect of insulin and glucose on the beta cells of the pancreatic islets in a fresh water teleost. Clurius butuuch44s. Acrn Zoo/. {Stockho/m) 50, 91-9.5. Khanna, S. S.. and Rekhari. K. (1972). Effect of glucose loading on the blood sugar and the histology of the pancreatic islets in a freshwater teleost, Heteropneustes fossilis. Actu Anat. 82, 126- 137. Larsson, A., and Lewander, K. (1972). Effects of glucagon administration to eels (Anguillu unguillu L.). Comp. Biochem. Physiol. 43, 831836. Mazeaud, F. ( 1973). Recherches sur la rigulation des acides gras libres plasmatique et de la glyckmie chez les poissons. PhD thesis. Faculty of Sciences, Paris. Patent, G. J., and FOB, P. P. (1971). Radioimmunoassay of insulin in fishes, experiments in viva and in vitro. Gen. Camp. Endocrinol. 16, 41-46. Tashima, L., and Cahill, G. F., Jr. (1968). Effects of insulin in the toadfish, Opsanus tuu. Gen. Camp. Endocrinol. 11, 262-271. Thorpe, A., and Ince, B. W. (1976). Plasma insulin levels in teleosts determined by a charcoalseparation radioimmunoassay technique. Gen. Coma. Endocrinol. 30. 332-339. Yalow, i. S., and Berson,‘S. A. (1965). Dynamics of insulin secretion in hypoglycaemia. Diabetes 14, 34 I -349.
AND
COMPARATIVE
ENDOCRINOLOGY
31,249-256
(1977)
Glucose and Amino Acid-Stimulated Insulin Release in Vivo in the European Silver Eel (Anguilla anguilla L.) BERNARD W. INCE AND ALAN THORPE Department
of Zoology & Comparative Physiology, Queen (University of London). Mile End Road, London, El 4NS, United Kingdom
Mary
College
Accepted October 22, 1976 Glucose and amino acid-stimulated insulin release was studied in vivo in cannulated European silver eels (Anguilla anguilla). Glucose-stimulated insulin release was dose dependent over a range of glucose loads (lo-100 mg/kg) while higher doses (300 and 500 mg/kg) produced no greater increments than 100 mg/kg. Arginine and lysine injections of 10, 25 and 100 mg/kg caused greater significant increases in plasma insulin levels than glucose at the same dose levels. Histidine (10 and 25 mglkg) caused a small but significant reduction in the plasma insulin level. Simultaneous injection of arginine (100 mglkg) and glucose (100 mgtkg) caused an increase in plasma insulin which was sustained for 60 min and remained significantly above the controls over the 360 min sampling period. Total insulin secretion appeared to be significantly greater over the entire sampling period than when arginine and glucose were injected alone.
In mammals and man, the factors which stimulate, modulate or inhibit insulin secretion have been studied in great detail both in viva and in vitro (cf. Gerich et al., 1976). In fishes, the few reports deal mainly with nutritional factors affecting insulin secretion. Thus, using a morphological approach it has been shown that glucose loading in certain fish species causes degranulation of the B-cells, implying glucose-stimulated insulin release (Khanna and Mehrotra, 1969; Khanna and Rekhari, 1972; Khanna and Gill, 1973). More quantitative data have been obtained by the use of radioimmunoassay of endogenous insulin levels. Tashima and Cahill (1968) observed that the serum insulin level of protein-fed toadfish, Opsanus tau, was significantly higher than in starved fish while oral glucose administration gave no response. Similar opposing effects of feeding and starvation on insulin levels have been observed more recently in goldfish, Carassius auratus (Patent and FOB, 1971), and in cod, Gadus morhua, and
rainbow trout, Salmo gairdneri (Thorpe and Ince, 1976). Patent and FOB (1971) reported in addition, that glucose and leucine, but not arginine, stimulate insulin release from the islets of toadfish in vitro and that the leucine effect is enhanced by the simultaneous addition of glucose. In the present investigation, the effects of glucose, arginine, lysine and histidine on plasma immunoreactive insulin levels have been studied in vivo in cannulated European silver eels, Anguilfa anguilla. MATERIALS
Maintenance Technique
AND METHODS
of Fish and Cannulation
European silver eels (A. anguilla) were purchased from a local dealer and maintained in polyethylene tanks supplied with a constant flow of fresh, aerated water. The mean weight of the eels used was 432 g (range 300-510 g). The experiments were performed between the period February to May 1976 when the tank water temperatures ranged from 9 to 13°C. Eels used in the present study had been fasting for 2-3 months prior to purchase and remained in this state
249 Copyright All rights
@ 1917 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
CtOl6-6480
250
INCE
AND
for the duration of the experiments. Prior histological investigation in such specimens has shown no evidence for degeneration of the endocrine pancreas (Ince and Thorpe, 1974). After a 2 day acclimation period, the fish were cannulated as described previously (Ince and Thorpe, 1974) and left for a further 3 days to allow for recovery from the stress effects caused by the operation (Thorpe and Ince, 1976). There were no significant variations in plasma glucose, amino acid nitrogen or insulin as related either to body weight or the duration of the period of this study.
Analytical
THORPE
levels of 10, 25 and 100 mgikg body weight and L-histidine at dose levels of 10 and 25 mg/kg body weight. It was not possible to use higher dose levels of histidine since these produced immediate and obvious signs of distress.
Statistical
Analysis
Paired t tests were used to test the significance between the means of the two preinjection control values and each of the values obtained following glucose or amino acid administration. The differences were considered significant when P < 0.05.
RESULTS
Techniques
Effects of Serial Blood Sampling on Plasma Glucose. Amino Acid Nitrogen and Insulin Levels Figure 1 shows that serial blood sampling does not induce any significant fluctuations in the levels of plasma glucose, amino acid nitrogen or insulin over a 360 min period. The control plasma insulin levels obtained Glucose and Amino Acid Administration in the present study, however, ranged from and Blood Sampling 2.40 to 3.80 rig/ml which is approximately Glucose and amino acids were dissolved in 2-3 times greater than those reported earphosphate-buffered. heparinised saline (pH 7) and lier from a study carried out between Ocadministered as single, rapid intra-arterial injections tober 1975 and January 1976; 1.12 + 0.10 through the cannula at varying dose levels. Blood rig/ml (11 animals) (Thorpe and Ince, 19761. samples (0.45 ml) were removed after 15. 30, 45, 60, Similarly, control plasma glucose levels 120, 180 and 360 min and a corresponding volume of were approximately 20-30 mg/dl greater buffer was replaced. Control levels of plasma glucose, amino acid nitrogen and insulin were assayed in than those previously reported, 42.2 t 5.0 two blood samples taken at 60 and 0 min prior to the mg/dl ( 11). It is not easy to ascribe precise injection of glucose or amino acids. D-Glucose was administered at dose levels of 10. reasons for these differences since assay techniques, blood sampling method and 25, 50, 100, 300 and 500 mglkg body weight. conditions were identical in L-Arginine and L-lysine were administered at dose experimental Plasma glucose, amino acid nitrogen and insulin were assayed in duplicate from a single plasma sample taken at each time interval from individual fish. The assays were performed either immediately or within 2 days from samples stored at -20°C. Details of the assay methods used have been given in earlier publications (Ince and Thorpe, 1974; Thorpe and Ince, 1976).
16) t
$I $GJ
1 k-H--: 0l5304560
(6)
t 120 time (minutes)
1 I 180
-_i 360
FIG. I. Effects of serial blood sampling on plasma glucose, amino acid nitrogen and insulin levels. Mean ‘t SE; number of animals in parentheses. The results show that serial blood sampling in cannulated silver eels over 360 min does not alter significantly the levels of plasma glucose, amino acid nitrogen and insulin.
INSULIN
RELEASE
IN
2.51
EELS
silver eels obtained from various localities around the British Isles.
the two studies. It may be that seasonal influences have a bearing on plasma glucase and insulin levels even in a naturally fasting animal such as the silver eel, but the differences could equally be of an intraspecific nature (Mazeaud, 1973), since we are dependent upon a commercial supply of
Effects of Intra-arterial on Plasma Glucose
Glucose Loading and Insulin Levels
Figures 2a and 2b show the response curves for plasma glucose and insulin given
11 d
1 -60
0
1.5
30
45
120
60 minutes
l&O
360
port-injection
FIG. 2a. Effects of intra-arterial glucose loading on plasma glucose and insulin levels. Mean ? SE; number of animals in parentheses; (0, A, W) glucose loads of IO, 25 and 50 mg/kg, respectively. The relationship between glucose levels and plasma insulin response is evident. Maximal insulin levels occur 30 min after injection and are significantly different from the controls. 900 ,700. e P -500. 4 s yoo 100. Oi
I
-60
0
15 30 45 60
120 minutes
180
360
port-injection
FIG. 2b. Effects of intra-arterial glucose loading on plasma glucose and insulin levels. Mean _CSE; number of animals in parentheses; (0, & 0) glucose loads of 100, 300 and 500 mgikg respectively. A significant elevation of plasma insulin occurring after 30 min is seen at 100 mg/kg, while at higher glucose doses, the elevation of insulin above control levels, although no greater than at 100 mg/kg, occurs after only I5 min. Recovery of plasma insulin to near-control levels occurs after 360 min at all doses despite the significantly elevated plasma glucose levels at this time
2.52
INCE
AND
by lo,25 and 50 mg/kg and 100,300 and 500 mg/kg glucose loads, respectively. At 10, 25, 50 and 100 mg/kg the mean maximal increases in plasma insulin above control levels after 30 min were 0.90, 1.51, 2.17, and 3.40 rig/ml, respectively. For glucose loads of 300 and 500 mglkg the mean maximal increases were 3.70 and 3.00 rig/ml, respectively, after only 15 min. These increased plasma insulin concentrations were significant at all dose levels but a log-linear relationship between dose of glucose and peak insulin values was observed only up to 50 mg/kg. At all dose levels of glucose, insulin returned to control values within the 360 min period despite the fact that glucose levels remained elevated at loads greater than 25 mg/kg.
THORPE
hyperglycaemia of 12 mgidl above control values but was without effect on plasma glucose at dose levels of 10 and 25 mg/kg. Effects of Intra-arterial Histidine Loading on Plasma Glucose, Amino Acid Nitrogen and Insulin Levels Histidine injections of 10 and 25 mg/kg caused a small but significant mean reduction in plasma insulin levels of between 0.70 and 0.93 rig/ml (Fig. 5). The response was slow, reaching a nadir between 60 and 120 min after injection with gradual recovery to control levels thereafter. No significant alterations in plasma glucose concentrations were observed at either dose level and only a small increase in amino acid nitrogen was seen after 15 min at 2.5 mg/kg.
Effects of Intra-arterial Arginine Loading Effects of Simultaneous Intra-arterial on Plasma Glucose, Amino Acid Arginine and Glucose Loading on Nitrogen and Insulin Levels Plasma Glucose, Amino Acid Nitrogen and Insulin Levels Arginine injections of 10, 25 and 100 Simultaneous administration of arginine mg/kg caused significant mean increases in plasma insulin above control levels of 1.67, ( 100 mg/kg) and glucose ( 100 mg/kg) caused 3.12 and 6.25 ngiml, respectively, after 15 a significant and sustained elevation of min (Fig. 3). Subsequent recovery of plasma insulin of 12.00 &ml above control plasma insulin to control levels was pre- levels between 15 and 60 min after injection ceded by that of amino acid nitrogen at 25 (Fig. 6). The mean sum of increments over and 100 mg/kg. A significant although tran- the entire sampling period was significantly sient hyperglycaemia of 10 mg/dl above greater than the summed insulin response control values occurred with arginine at 100 given by arginine or glucose alone over the mg/kg, while no significant effects on same period. Between 60 and 360 min after injection, plasma insulin levels fell slowly plasma glucose were observed at the lower dose levels. by a total of only 3.55 rig/ml so that after 360 min the plasma insulin levels were still Effects of Intra-arterial Lysine Loading on significantly greater than the controls. The Plasma Glucose, Amino Acid Nitrogen simultaneous injection of arginine and gluand Insulin Levels cose was followed by a significantly accelerated clearance of the excess glucose Lysine injections of IO,25 and 100 mg/kg when compared with the clearance of glucaused significant mean increases in plasma insulin above control levels of 2.43, 5.35 cose from plasma following a single glucose and 10.86 rig/ml, respectively, after 15 min load of 100 mg/kg. The plasma clearance of amino acid nitrogen, however, was not sig(Fig. 4) with the plasma insulin clearance closely resembling that for amino acid ni- nificantly altered by a simultaneous injectrogen at all dose levels. Lysine at 100 tion of arginine and glucose when compared with arginine alone. mg/kg caused a significant but transient
INSULIN
RELEASE
IN
253
EELS
360 minutes
post-
injection
FIG. 3. Effects of intraarterial arginine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean f SE; number of animals in parentheses; (0, A, I) Arginine loads of 10, 25 and 100 &kg, respectively. Arginine at all doses causes significant maximal elevation of plasma insulin after 15 min. At 100 mg/kg, however, a reduced rate of plasma insulin clearance is seen when compared with that of amino acid nitrogen. A significant but transient hyperglycaemia occurs with an arginine load of 100 mg/kg.
DISCUSSION pling frequency used in the present study. The present data show that glucose, ar- Thus, the increased rate of fall of plasma ginine and lysine stimulate insulin release in glucose which follows the higher loads (Fig. viva in silver eels and that the initial rise in 2b) cannot be due to insulin alone. As has been shown previously (Ince and Thorpe, plasma insulin levels following the administration of the amino acids is greater than for 1974), the terminal portion of the glucose loading curve (assimilation phase) shows glucose at the same dose level. only a slow recovery to initial levels and The glucose loading data indicate a direct relationship between increasing glucose from the present data it can be seen that load and the rate of fall of plasma glucose during this time insulin levels are falling during the initial 30 min period. At this correspondingly. Data from mammalian studies show that time, over the range lo-100 mg/kg, there is a corresponding increase in the concen- certain amino acids stimulate not only the tration of plasma insulin which could ac- release of insulin but also growth hormone count for the accelerated removal of the and glucagon (cf. Gerich er al., 1976). Since excess glucose from the blood. The level of the latter hormones are also known to plasma insulin attained after a glucose load stimulate insulin secretion, the effect of inof 100 mg/kg was not seen to be signifi- jected arginine and lysine in the present study (Figs. 3 and 4) may be due to a comcantly exceeded if the dose was increased to 300 or 500 mg/kg, at least with the sam- bination of secondary hormonal influences
254
INCE
minutes
AND
THORPE
post-injectbn
FIG. 4. Effects of intra-arterial lysine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean f SE; number of animals in parentheses; symbols and dose levels as in Fig. 3. Lysine at all doses causes a significant maximal elevation of plasma insulin after 15 min. A significant but transient hyperglycaemia occurs with lysine at 100 mg/kg.
FIG. 5. Effects of intra-arterial histidine loading on plasma insulin levels. Mean + SE; number of animaIs in parentheses; (0, A) histidine loads of 10 and 25 mgikg, respectively. Histidine at both doses causes a significant but transient reduction in plasma insulin.
INSULIN
RELEASE
IN
255
EELS
1
-60
0
15 30 45 60 minutes
120 port-injeciion
180
360
FIG. 6. Effects of simultaneous intraarterial glucose and arginine loading on plasma glucose, amino acid nitrogen and insulin levels. Mean 2 SE; number of animals in parentheses; (0) simultaneous injection of arginine (100 mg/kg) and glucose (100 mg/kg). The effects of glucose (0) and arginine alone (m), shown earlier, are given here for comparison. Simultaneous injection of arginine and glucose results in a significant elevation of plasma insulin over the entire sampling period. The total insulin secretion appears significantly greater than when either is injected alone. Plasma clearance of amino acid nitrogen remains unchanged but excess glucose removal is accelerated.
and a direct interaction of the amino acids with the B-cells. In addition, the arginine and lysine-induced hyperglycaemia, seen at 100 mglkg, results presumably from the influence of released glucagon which is known to be glycogenolytic in eels (Larsson and Lewander, 1972; Ince, 1975). By comparison with the present data, Patent and FoA (1971) reported that both glucose and leucine, but not arginine, caused significant insulin release from the islets of toadfish, 0. tau in vitro, whereas oral glucose loading in the same species failed to elicit insulin release (Tashima and Cahill, 1968). Patent and FOB (1971) also reported a synergistic effect of glucose and leucine on insulin release from toadfish islets in vitro. Likewise in the present study, the simul-
taneous injection of arginine and glucose appeared to cause a significantly greater total insulin secretion over the entire sampling period than when either was injected alone (Fig. 6). However, this apparent synergism between glucose and arginine in vivo may have been caused by concomitant release of glucagon (or other hormones) stimulating the B-cells as well as by the direct effects of glucose and arginine. It is interesting to note that in silver eels, histidine does not stimulate insulin release but in fact causes a reduction in plasma insulin levels (Fig. 5). Similarly, in man, histidine has been shown to be the least potent of the amino acids in stimulating insulin release (Fajans and Floyd, 1972). The reasons for the marked difference in its ac-
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tions when compared with other amino acids such as arginine and lysine, are unknown. The plasma clearance of arginine and lysine as judged by total plasma amino acid nitrogen is very similar at all dose levels (Figs. 3 and 4). It is of interest, therefore, that the shape of the plasma insulin curves following their injection differs markedly. It is likely that the sustained elevation of plasma insulin following arginine injection, either alone or with glucose, is due to a greater total output of the hormone caused by a different response of the B-cells to that following lysine injection. Alternatively, it could be argued that the differences between the curves are due either to insulin distribution and degradation phenomena as has been emphasised by Yalow and Berson (1965) or to secondary hormonal influences on the B-cell arising from the initial amino acid stimulus. The latter idea is supported by the observation that hypoglycaemia during pronounced and prolonged hyperinsulinaemia is absent, suggesting that glucagon or other insulin antagonists may be operating to maintain plasma glucose at a normal level. ACKNOWLEDGMENT This investigation forms part of a research project supported by a grant to one of us (A. T.) from the Science Research Council, England, to whom our thanks are due.
REFERENCES Fajans, S. S., and Floyd, J. C., Jr. (1972). Stimulation of islet cell secretion by nutrients and by gastrointestinal hormones released during digestion.
In “Handbook of Physiology” (D. F. Steiner and N. Freinkel, eds.). Vol. 1, Sect. 7. pp. 473-493. Amer. Physiol. Sot., Washington, D.C. Gerich, J. E., Charles, M. A., and Grodsky, G. M. (1976). Regulation of pancreatic insulin and glucagon secretion. Ann. Rev. Physiol. 38, 353388. Ince, B. W. (1975). Studies on the hormonal control of metabolism in teleosts. PhD thesis, University of London, England. Ince, B. W.. and Thorpe, A. (1974). Effects of insulin and of metabolite loading on blood metabolites in the European silver eel (Anguillu ungttilh L.). Gen. Comp. Endocrinol. 23, 460-471. Khanna, S. S., and Gill. T. S. (1973). Effect of glucose loading on the blood glucose level and histology of the principal islets in Channa puncfatus. Endocrinology (Japan) 20, 375-383. Khanna, S. S., and Mehrotra. B. K. (1969). Effect of insulin and glucose on the beta cells of the pancreatic islets in a fresh water teleost. Clurius butuuch44s. Acrn Zoo/. {Stockho/m) 50, 91-9.5. Khanna, S. S.. and Rekhari. K. (1972). Effect of glucose loading on the blood sugar and the histology of the pancreatic islets in a freshwater teleost, Heteropneustes fossilis. Actu Anat. 82, 126- 137. Larsson, A., and Lewander, K. (1972). Effects of glucagon administration to eels (Anguillu unguillu L.). Comp. Biochem. Physiol. 43, 831836. Mazeaud, F. ( 1973). Recherches sur la rigulation des acides gras libres plasmatique et de la glyckmie chez les poissons. PhD thesis. Faculty of Sciences, Paris. Patent, G. J., and FOB, P. P. (1971). Radioimmunoassay of insulin in fishes, experiments in viva and in vitro. Gen. Camp. Endocrinol. 16, 41-46. Tashima, L., and Cahill, G. F., Jr. (1968). Effects of insulin in the toadfish, Opsanus tuu. Gen. Camp. Endocrinol. 11, 262-271. Thorpe, A., and Ince, B. W. (1976). Plasma insulin levels in teleosts determined by a charcoalseparation radioimmunoassay technique. Gen. Coma. Endocrinol. 30. 332-339. Yalow, i. S., and Berson,‘S. A. (1965). Dynamics of insulin secretion in hypoglycaemia. Diabetes 14, 34 I -349.
