Dose-Response Studies on the Inhibitory Effect of Thyroid Hormones on Insulin Secretion in the Rat Sigurd Dose-response studies have been performed to investigate the effect of thyroid hormones on insulin secretion from the rat pancreas with special reference to the time course of the hormone effect, doses of triiodothyronine (Ts) and thyroxine (T,), and glucose concentration in the perfusion medium. The prominent effect of thyroid hormones was the inhibition of the late phase of glucose-induced insulin secretion. As the late phase comprises at least 98%-99% of insulin released from the pancreas during a 60-min stimulation period with glucose, 60-min cumulative
Lenzen values were calculated. Both Ts and T, inhibited insulin secretion and induced concomitant inhibitory effects on plasma cholesterol levels, a parameter of experimental hyperthyroidism. The correlation demonstrates that inhibition of glucoseinduced insulin secretion from the pancreas is a specific effect of thyroid hormones. The inhibitory effect of Ts was five times greater than the inhibitory effect of T,. An excess of thyroid hormones induced hyperthyroidism, with its well-known increased incidence of diabetes.
T
HE DIABETIC POTENCY of thyroid hormones has been known for many years.’ Studies on the effects of thyroid hormones on insulin secretion, however, have provided conflicting results (for discussion, see Lenzen et al.*). Due to the bewildering array of differences in experimental design and techniques conclusive explanations could not be provided. In a recent communication* we showed that experimental hyperthyroidism after administration of thyroid hormones in vivo inhibited insulin secretion from the perfused rat pancreas, whereas experimental hypothyroidism was accompanied by increased insulin secretion. Thyroid hormones do not inhibit insulin secretion directly when administered in vitro (S. Lenzen, unpublished results).3 Dose-response relationships have never been studied. Therefore, dose-response studies were performed. The effects of three variables-the time course of the hormone effect, doses of triodothyronine (T,) and thyroxine (T4), and glucose concentration in the perfusion mediumon insulin secretion from the perfused rat pancreas were investigated. These dose-response studies provide a basis for studies on the cellular events that accompany the inhibitory effect of thyroid hormones on insulin secretion. In combination with results presented earlier:5 the present studies allow a precise characterization of insulin secretion in thyroid hormone diabetes. Thyroid hormones decrease glucose tolerance, ’ increase blood glucose levels in vivo,2*4and decrease glucose-induced insulin secretion in animals,2~5 mimicking essential features of human nonobese maturity-onset diabetes. Therefore, exFrom the Inslirute of Pharmacology and Toxicology. University of GGttingen. .Receivedfor publication March 23, 1977. Supported by the Deutsche Forschungsgemeinschaft.
Gbttingen,
Germany.
Reprint requests should be addressed to Dr. Sigurd Lenren, Institur ftir Pharmakologie : Toxikologie. Robert-Koch-.%. 40. D-3400 Gijtringen. Germany. 0 1978 by Grune & Stratton. Inc. ISSN 0026-0495. 00260495/78/2701-0010$02.00/0
Metabolism,
Vol.
27,
No.
1 (January),
1978
und
81
82
SIGURD
perimental animals.
hyperthyroidism
is an interesting
MATERIALS
diabetes
model
for
LENZEN
studies
in
AND METHODS
Chemicals Pure rat insulin was kindly supplied by Novo, from
Serva,
‘251-labeled
Heidelberg,
L-triiodothyronine
bovine insulin from Behringwerke
grade, including
D-glucose,
Mainz:
bovine albumin
and L-thyroxine
were from Merck
AG. AG,
from
Frankfurt.
(fraction
Sigma,
All other
V) was obtained
St. Louis,
chemicals
MO.,
and
of analytical
Darmstadt.
Experimental Design Experiments
were performed
on age- and weight-paired
weight. T3, T4, or an equivalent
volume
(up to 20 g) after thyroid hormone
administration
The pancreas
and the adjacent
i.p.
rats of 200-270
Reduced
g body
body weight
gain
was dose dependent.
part of the duodenum,
moved from 24-hr fasted rats according
male Wistar
of saline was injected
to the method
the spleen, of Grodsky
and the stomach
were re-
et al.6 with slight moditica-
tions.4
Analytical Methods Immunoreactive Plasma cholesterol test combination,
insulin in the perfusate determinations
courtesy of Boehringer
method requires only small amounts
was determined
were performed Mannheim
by the method
of Zaharko
with a new enzymatic GmbH,
Mannheim).
method’
Determination
of plasma and can easily be performed
and Beck.’ (Boehringer with this
in a large number
of
one-way
of
samples.*
Calculations Results
were tested for statistical
significance
with Student’s
t
test and
analysis
variance.
RESULTS
The effect of thyroid hormones on insulin secretion with special reference to the time course of the hormone effect, doses of T, and T4, and glucose concentration in the perfusion medium was investigated. T3 as well as T4 had a slight stimulatory effect on the immediate glucoseinduced (16.7-33.4 mM) insulin secretory response of the perfused rat pancreas, as shown before for single doses of Tj and T4 (see Fig. 1 in Lenzen et a1.2*4*5*). This effect was preferentially seen in the intermediate dose range. Even the lowest glucose concentration in the perfusion medium (8.3 mM) tested induced a high immediate insulin secretory response in controls not significantly different from those induced by higher glucose concentrations (16.7-33.4 mM). In the pancreas obtained from rats treated with T3 (200 rg/kg/day for 5 days) or with T4 (1000 pg/kg/day for 5 days), however, 8.3 mM glucose provoked no immediate insulin secretory response. The prominent effect of thyroid hormones in all dose-response studies was the inhibition of the late phase of glucose-induced insulin secretion. As the late phase of glucose-induced insulin secretion comprises at least 98”,-99”, of insulin released from the perfused pancreas during a 60-min perfusion period,’ 60-min cumulative values were calculated. They will virtually represent latephase insulin secretion in all figures. Decreased plasma cholesterol levels proved to be the best parameter of experimental hyperthyroidism in a previous study.*
TH AND INSULIN SECRETION
Fig. 1. Time course of the inhibitory effeet of a single dose of T3 (2 mg/kg) (e) on plasma cholesterol levels ( p < 0.05) (upper curve) and glucose-induced (16.7 mM) inrulin secretion (lower curve) from the perfused rat pcmcreor ( p < O.OOl), compared with control (0). Mean + SEM; 3-8 experiments in each group.
z! L3. z ; 2t p
l-
.$ o
L 01234567
days atfer
lriiodolhyronine
I4
administration
An inhibition of glucose-induced insulin secretion from the perfused rat pancreas always went along with decreased plasma cholesterol levels (Figs. l-4, Table 1). A single injection of TJ (2 mg/kg) caused an inhibition of glucose-induced (16.7 mM) insulin secretion 2-4 days after administration, with a maximal inhibitory effect after 3 days (Fig. 1) (p < 0.001). While Tj induced only a relatively short inhibition (2-3 days), T.,-mediated inhibition was evident for 8-12 days (Fig. 2). A single injection of T, (20 mg/kg) resulted in an inhibition of glucose-induced (I 6.7 mM) insulin secretion 2- I2 days after administration, with a maximal inhibitory effect after 5-8 days (Fig. 2) (p < 0.001). The inhibitory effect of T3 and T4 disappeared again and insulin secretion returned to normal levels (Figs. 1 and 2). Slightly increased insulin secretion 3-4 wk after T4 administration is presumably due to a body weight gain in the donor rats and hence increased insulin secretory capacity of the perfused pancreas several weeks after the injection, and not due to any specific effect of T4 (Fig. 2). The
Fig. 2. Time course of the inhibitory effect of Q single dose of T4 (20 mg/kg) (e) on plasma cholesterol levels ( p < 0.05) (upper curve) ond glucose-induced (16.7 mm) insulin sew&ion (lower curve) from the perfused ret pancreas ( p < 0.001). compared with control (0). Mean f SEM; 4-8 experiments in each group.
SIGURD LENZEN
.s 1. 3 .s
o- 1 0
200
dose
400
of lriodothyronine
600
(Mg/kg)
Fig. 3. Dose-depndent inhibitory effect of T3 ( 10-600 rg/kg/day for 5 days) ( l) on plasma cholesterol levels ( p < 0.001) (upper curve) and glucose-induced (16.7 mm) insulin secretion (lower curve) from the perfused rat pancreas (p < 0.05), compared with control (0). Mean f SEM; 4-8 experiments in each group.
kinetics, the decrease, and the subsequent increase of plasma cholesterol levels as a parameter of experimental hyperthyroidism were similar to the pattern of insulin secretion under the influence of thyroid hormones; but the effects of T3 as well as of T4 on insulin secretion followed the effects on plasma cholesterol levels with a short delay (Figs. 1 and 2) (p < 0.05). For the following experiments a S-day period of daily hormone treatment was chosen on the basis of the results obtained in Figs. 1 and 2. A 5-day period of treatment with T3 was long enough to maintain a permanent and reproducible inhibition of insulin secretion in all experiments, while it was the shortest possible period to obtain a reliable and constant inhibition of insulin secretion by T4 treatment. Both T3 (lo-600 rg/kg/day for 5 days) and T4 (50-2000 pg/kg/day, for 5 days) had a dose-dependent and exclusively inhibitory effect on glucose-induced
fig. 4. Dose-dependent inhibitory effect of T4 (50-2000 pg/kg/day for 5 days) (e) on plasma cholesterol levels (p < 0.001) (upper curve) and glucose-induced (16.7 mm) insulin secretion (lower curve) from the perfused rat pancreas (p < 0.05). compared with control (0). Mean f SEM; 4-8 experiments in each group.
.C, I 3 g ._ 0-
0 0
500
1000 1500 dose of thyroxine
2000 (Pg/kg)
TH AND INSULIN
85
SECRETION
97
8. 76. **
Effect of 13 (200 &kg/day for Fig. 5. 5 days) (e) in comparison with controls (0) on insulin secretion from the rat pancreas in relation to glucose concentration (8.3-33.4 mM) in the perfusion medium. Mean f SEM; 3-8 experiments in each group. l lp < compared with con0.01, **+p < 0.001,
8.3
trols (0).
16.7 25.0 glucose concenlrotion
33.4
(m&l)
(16.7 mM) insulin secretion (p < 0.05) and decreased plasma cholesterol levels over a wide range of doses tested (Figs. 3 and 4) (p < 0.001). Tj (Fig. 3) was five times more potent than T4 (Fig. 4). There was a sigmoidal relationship between insulin secretion from the perfused pancreas and glucose concentration in the perfusion medium (Figs. 5 and 6). T3 (200 pg/kg/day for 5 days) and T4 (1000 pg/kg/day, for 5 days) had the same hyperthyroid potency, as shown by their inhibitory effect on plasma cholesterol (Table 1). Both TS and T., had identical inhibitory effects on insulin secretion from the pancreas in relation to glucose concentration in the perfusion medium (Figs. 5 and 6). In the presence of 8.3 mM glucose in the perfusion medium insulin secretion was not inhibited because 60-min cumulative insulin secretion values (Figs. 5 and 6) represent mainly the inhibition of
62 ‘C 5. E 0 e49 L3!i
*
$2.c, 1.
3
20.
0
8.3
16.7 25.0 glucose concentration
33.4
(mM)
Fig. 6. Effect of T4 (1000 &kg/day for 5 days) (e) in comparison with controls (0) on insulin secretion from the rat pancreas in relation to glucose concentration (8.3-33.4 mm) in the perfusion medium. Mean f SEM; 4-8 experiments in each group. lp < 0.05; l**p < 0.001 compared with controls (0).
86
SIGURD
Table 1. Effect of T3 (200 &kg/day Equivalent
Volume of Saline (Control)
Treatment
for 5 days), T, (1000 on Plasma Cholesterol ”
pg/kg/day
for 5 days), or an
levels in Rats (Mean f
19
54.9
T3
12
41 .o * 3.9*
16
41.2
TA
SEM)
Plasma Cholesterol (mg/lOO ml)
Control
*p < 0.01 compared
LENZEN
f
2.5
* 3.3*
with control.
the late phase of glucose-induced insulin secretion,’ and the late phase of secretion is not yet present at such a low glucose concentration.4 At higher glucose concentrations (16.7,25.0, and 33.4 mM) in the perfusion medium insulin secretion was significantly inhibited after treatment of the rats with Tj as well as T4 due to their effect on the late phase of secretion. Figures 5 and 6 indicate that the K, of the sigmoidal concentration-response curve was not affected, whereas V,,,,, was decreased (Figs. 5 and 6). DISCUSSION
The inhibitory effect of thyroid hormones on insulin secretion can be characterized as follows: (1) The speed of onset of the inhibitory effect of Tj is more rapid and the time required to develop its maximal inhibitory effect is shorter, whereas the duration of the inhibitory effect of T4 is longer and weeks are needed for complete regression of the inhibitory effect of T4 (Figs. 1 and 2). (2) The inhibitory effects of TJ and T4 are dose dependent (Figs. 3 and 4). (3) TJ is five times more potent than T4 (Figs. 3-6). The effects of T3 as well as T4 on plasma cholesterol levels as a parameter of the hyperthyroid effect correlate with those on insulin secretion, supporting the conclusion drawn from earlier experiments’ that inhibition of glucoseinduced insulin secretion is a specific effect of thyroid hormones. None of the different experimental situations provided evidence for a stimulatory effect of thyroid hormones on glucose-induced insulin secretion, though this has been often supposed (for discussion, see Lenzen et al.‘). At present I can provide no explanation for the more rapid inhibitory effect of thyroid hormones on plasma cholesterol levels, the only discrepancy between the effect of thyroid hormones on cholesterol levels and insulin secretion. The different pharmacokinetic characteristics and the generally known greater biologic potency of Tj in comparison to T4 are unexplained phenomena.” Two alternatives are being considered at present and both have to be considered as possible explanations: ” (I) T4 may be a prohormone that must be converted to Tj to exert hormonal effects; or (2) the great differences in T3- and T,-binding properties on either side of the cell membrane may explain these findings. The fact that thyroid hormones that are diabetogenic in animals and in man’ can induce a state of hyperthyroidism in animals as well as in man makes it possible to draw conclusions that may help to improve the understanding of diabetes in man. Such conclusions cannot be drawn from studies on animals with genetic or chemical diabetes because such states cannot be reproduced in man. The present dose-response studies (Figs. l-6) together with results obtained
TH AND INSULIN
a7
SECRETION
in earlier studies2*4*5allow the characterization of experimental thyroid diabetes in animals as an experimental diabetes model. All the features are also known to be typical characteristics of human nonobese maturity-onset diabetes:‘**‘3 (1) selective and dose-dependent inhibition of glucose-induced insulin secretion of tolbutamide-, glyceraldehyde-, and (Figs. 3 and 4); 4~‘2(2) no inhibition mannose-induced insulin secretion;4’5”2 (3) absence of an immediate insulin secretory response of the pancreas from thyroid hormone-treated rats in response to a low glucose stimulus (8.3 mM) in contrast to a significant response in controls, as described in the results section’2*‘3 (the reason for this phenomenon is apparently the fact that an increase in the glucose concentration of the perfusion medium from 0 to 8.3 mM glucose was not sufficient to induce an immediate insulin secretory response of the pancreas from rats treated with T3 or T4 because these animals had increased plasma glucose levels in vivo already.2*4 In the presence of higher glucose concentrations (16.7-33.4 mM) in the perfusion medium the immediate insulin secretory response of the pancreas was not inhibited by T3 or T4 treatment of the rats); (4) selective inhibition of the late phase of glucose-induced insulin secretion (Figs. 3 and 4);4*5S’2*‘3 (5) reduction of the inhibition of glucose-induced (16.7 mM) insulin secretion after preperfusion of the pancreas with a substimulatory glucose concentration (5.5 mM) (unpublished observation);‘* (6) reduction of the relative inhibition of insulin secretion in the presence of a high stimulatory glucose concentration (33.4 mM) (Figs. 5 and 6);5 and (7) reduction of the relative inhibition of insulin secretion after feeding of the rats before the experiments.5.‘2 Two conclusions can be drawn from the experiments: (1) Inhibition of glucose-induced insulin secretion from the perfused pancreas is a specific effect of thyroid hormones characterized by features that are also known to be typical for nonobese maturity-onset diabetes in man. (2) Experimental hyperthyroidism in animals and hyperthyroidism in man have similar characteristics.‘** These similarities make it possible to estimate the relevance of results obtained in animal studies on thyroid diabetes for the understanding of diabetes in man. ACKNOWLEDGMENT The skillful technical
assistance
of S. Detels and R. LotTIer is gratefully
acknowledged.
REFERENCES I. Houssay BA: The action of the thyroid on diabetes. Recent Prog Horm Res 2:277, 1948 2. Lenzen S. Joost HG. Hasselblatt A: Thyroid function and insulin secretion from the perfused pancreas in the rat. Endocrinology 99: 125, 1976 3. Malaisse WJ, Malaisse-Lagae F. McGraw EF: Effects of thyroid function upon insulin secretion. Diabetes 16:643, 1967 4. Lenzen S. Panten U. Hasselblatt A: Thyroxine treatment and insulin secretion in the rat. Diabetologia I l:49, 1975 5. Lenzen S, Joost HG, Hasselblatt A: The inhibition of insulin secretion from the perfused
rat pancreas after thyroxine treatment. Diabetologia 12:495, 1976 6. Grodsky GM. Batts AA, Bennett LL. Vcella C, McWilliams NB. Smith PF: EtTects of carbohydrates on secretion of insulin from isolated rat pancreas. Am J Physiol 205:638, 1963. 7. Zaharko DS, Beck LV: Studies of a simplified plasma insulin immunoassay using cellulose powder. Diabetes 17:444, 1968 8. Roschlau P, Bernt E, Gruber W: Enzymatische Bestimmung des Gesamt-Cholesterins im Serum. Z Klin Chem Klin Biochem 121226, 1974
88
9. Grodsky GM: A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling. J Clin Invest 51:2047, 1972 IO. Pittman CS, Pittman JA: Relation of chemical structure to the action and metabolism of thyroactive substances, in Greer MA, Solomon DH (eds): Handbook of Physiology. Section 7: Endocrinology, vol 3, Thyroid. Washington, DC, American Physiological Society, 1974, pp 233-253 1I. DiStefano JJ, Fisher DA: Peripheral
SIGURD LENZEN
distribution of the thyroid hormones. A priPharmacol marily quantitative assessment. Ther [B] 2:539, I976 12. Cerasi E: Insulin secretion: Mechanism of the stimulation by glucose. Rev Biophys 8:l. 1975 13. Schulz B, Michaelis D, Neumann 1. Gottschling D, Bibergeil H: Kohlenhydrattoleranz und Insulinverhalten im Glukoseinfusionstest bei normgewichtigen Personen mit Protodiabetesverdacht. Z Med 5: 138, 1976
Lenzen values were calculated. Both Ts and T, inhibited insulin secretion and induced concomitant inhibitory effects on plasma cholesterol levels, a parameter of experimental hyperthyroidism. The correlation demonstrates that inhibition of glucoseinduced insulin secretion from the pancreas is a specific effect of thyroid hormones. The inhibitory effect of Ts was five times greater than the inhibitory effect of T,. An excess of thyroid hormones induced hyperthyroidism, with its well-known increased incidence of diabetes.
T
HE DIABETIC POTENCY of thyroid hormones has been known for many years.’ Studies on the effects of thyroid hormones on insulin secretion, however, have provided conflicting results (for discussion, see Lenzen et al.*). Due to the bewildering array of differences in experimental design and techniques conclusive explanations could not be provided. In a recent communication* we showed that experimental hyperthyroidism after administration of thyroid hormones in vivo inhibited insulin secretion from the perfused rat pancreas, whereas experimental hypothyroidism was accompanied by increased insulin secretion. Thyroid hormones do not inhibit insulin secretion directly when administered in vitro (S. Lenzen, unpublished results).3 Dose-response relationships have never been studied. Therefore, dose-response studies were performed. The effects of three variables-the time course of the hormone effect, doses of triodothyronine (T,) and thyroxine (T4), and glucose concentration in the perfusion mediumon insulin secretion from the perfused rat pancreas were investigated. These dose-response studies provide a basis for studies on the cellular events that accompany the inhibitory effect of thyroid hormones on insulin secretion. In combination with results presented earlier:5 the present studies allow a precise characterization of insulin secretion in thyroid hormone diabetes. Thyroid hormones decrease glucose tolerance, ’ increase blood glucose levels in vivo,2*4and decrease glucose-induced insulin secretion in animals,2~5 mimicking essential features of human nonobese maturity-onset diabetes. Therefore, exFrom the Inslirute of Pharmacology and Toxicology. University of GGttingen. .Receivedfor publication March 23, 1977. Supported by the Deutsche Forschungsgemeinschaft.
Gbttingen,
Germany.
Reprint requests should be addressed to Dr. Sigurd Lenren, Institur ftir Pharmakologie : Toxikologie. Robert-Koch-.%. 40. D-3400 Gijtringen. Germany. 0 1978 by Grune & Stratton. Inc. ISSN 0026-0495. 00260495/78/2701-0010$02.00/0
Metabolism,
Vol.
27,
No.
1 (January),
1978
und
81
82
SIGURD
perimental animals.
hyperthyroidism
is an interesting
MATERIALS
diabetes
model
for
LENZEN
studies
in
AND METHODS
Chemicals Pure rat insulin was kindly supplied by Novo, from
Serva,
‘251-labeled
Heidelberg,
L-triiodothyronine
bovine insulin from Behringwerke
grade, including
D-glucose,
Mainz:
bovine albumin
and L-thyroxine
were from Merck
AG. AG,
from
Frankfurt.
(fraction
Sigma,
All other
V) was obtained
St. Louis,
chemicals
MO.,
and
of analytical
Darmstadt.
Experimental Design Experiments
were performed
on age- and weight-paired
weight. T3, T4, or an equivalent
volume
(up to 20 g) after thyroid hormone
administration
The pancreas
and the adjacent
i.p.
rats of 200-270
Reduced
g body
body weight
gain
was dose dependent.
part of the duodenum,
moved from 24-hr fasted rats according
male Wistar
of saline was injected
to the method
the spleen, of Grodsky
and the stomach
were re-
et al.6 with slight moditica-
tions.4
Analytical Methods Immunoreactive Plasma cholesterol test combination,
insulin in the perfusate determinations
courtesy of Boehringer
method requires only small amounts
was determined
were performed Mannheim
by the method
of Zaharko
with a new enzymatic GmbH,
Mannheim).
method’
Determination
of plasma and can easily be performed
and Beck.’ (Boehringer with this
in a large number
of
one-way
of
samples.*
Calculations Results
were tested for statistical
significance
with Student’s
t
test and
analysis
variance.
RESULTS
The effect of thyroid hormones on insulin secretion with special reference to the time course of the hormone effect, doses of T, and T4, and glucose concentration in the perfusion medium was investigated. T3 as well as T4 had a slight stimulatory effect on the immediate glucoseinduced (16.7-33.4 mM) insulin secretory response of the perfused rat pancreas, as shown before for single doses of Tj and T4 (see Fig. 1 in Lenzen et a1.2*4*5*). This effect was preferentially seen in the intermediate dose range. Even the lowest glucose concentration in the perfusion medium (8.3 mM) tested induced a high immediate insulin secretory response in controls not significantly different from those induced by higher glucose concentrations (16.7-33.4 mM). In the pancreas obtained from rats treated with T3 (200 rg/kg/day for 5 days) or with T4 (1000 pg/kg/day for 5 days), however, 8.3 mM glucose provoked no immediate insulin secretory response. The prominent effect of thyroid hormones in all dose-response studies was the inhibition of the late phase of glucose-induced insulin secretion. As the late phase of glucose-induced insulin secretion comprises at least 98”,-99”, of insulin released from the perfused pancreas during a 60-min perfusion period,’ 60-min cumulative values were calculated. They will virtually represent latephase insulin secretion in all figures. Decreased plasma cholesterol levels proved to be the best parameter of experimental hyperthyroidism in a previous study.*
TH AND INSULIN SECRETION
Fig. 1. Time course of the inhibitory effeet of a single dose of T3 (2 mg/kg) (e) on plasma cholesterol levels ( p < 0.05) (upper curve) and glucose-induced (16.7 mM) inrulin secretion (lower curve) from the perfused rat pcmcreor ( p < O.OOl), compared with control (0). Mean + SEM; 3-8 experiments in each group.
z! L3. z ; 2t p
l-
.$ o
L 01234567
days atfer
lriiodolhyronine
I4
administration
An inhibition of glucose-induced insulin secretion from the perfused rat pancreas always went along with decreased plasma cholesterol levels (Figs. l-4, Table 1). A single injection of TJ (2 mg/kg) caused an inhibition of glucose-induced (16.7 mM) insulin secretion 2-4 days after administration, with a maximal inhibitory effect after 3 days (Fig. 1) (p < 0.001). While Tj induced only a relatively short inhibition (2-3 days), T.,-mediated inhibition was evident for 8-12 days (Fig. 2). A single injection of T, (20 mg/kg) resulted in an inhibition of glucose-induced (I 6.7 mM) insulin secretion 2- I2 days after administration, with a maximal inhibitory effect after 5-8 days (Fig. 2) (p < 0.001). The inhibitory effect of T3 and T4 disappeared again and insulin secretion returned to normal levels (Figs. 1 and 2). Slightly increased insulin secretion 3-4 wk after T4 administration is presumably due to a body weight gain in the donor rats and hence increased insulin secretory capacity of the perfused pancreas several weeks after the injection, and not due to any specific effect of T4 (Fig. 2). The
Fig. 2. Time course of the inhibitory effect of Q single dose of T4 (20 mg/kg) (e) on plasma cholesterol levels ( p < 0.05) (upper curve) ond glucose-induced (16.7 mm) insulin sew&ion (lower curve) from the perfused ret pancreas ( p < 0.001). compared with control (0). Mean f SEM; 4-8 experiments in each group.
SIGURD LENZEN
.s 1. 3 .s
o- 1 0
200
dose
400
of lriodothyronine
600
(Mg/kg)
Fig. 3. Dose-depndent inhibitory effect of T3 ( 10-600 rg/kg/day for 5 days) ( l) on plasma cholesterol levels ( p < 0.001) (upper curve) and glucose-induced (16.7 mm) insulin secretion (lower curve) from the perfused rat pancreas (p < 0.05), compared with control (0). Mean f SEM; 4-8 experiments in each group.
kinetics, the decrease, and the subsequent increase of plasma cholesterol levels as a parameter of experimental hyperthyroidism were similar to the pattern of insulin secretion under the influence of thyroid hormones; but the effects of T3 as well as of T4 on insulin secretion followed the effects on plasma cholesterol levels with a short delay (Figs. 1 and 2) (p < 0.05). For the following experiments a S-day period of daily hormone treatment was chosen on the basis of the results obtained in Figs. 1 and 2. A 5-day period of treatment with T3 was long enough to maintain a permanent and reproducible inhibition of insulin secretion in all experiments, while it was the shortest possible period to obtain a reliable and constant inhibition of insulin secretion by T4 treatment. Both T3 (lo-600 rg/kg/day for 5 days) and T4 (50-2000 pg/kg/day, for 5 days) had a dose-dependent and exclusively inhibitory effect on glucose-induced
fig. 4. Dose-dependent inhibitory effect of T4 (50-2000 pg/kg/day for 5 days) (e) on plasma cholesterol levels (p < 0.001) (upper curve) and glucose-induced (16.7 mm) insulin secretion (lower curve) from the perfused rat pancreas (p < 0.05). compared with control (0). Mean f SEM; 4-8 experiments in each group.
.C, I 3 g ._ 0-
0 0
500
1000 1500 dose of thyroxine
2000 (Pg/kg)
TH AND INSULIN
85
SECRETION
97
8. 76. **
Effect of 13 (200 &kg/day for Fig. 5. 5 days) (e) in comparison with controls (0) on insulin secretion from the rat pancreas in relation to glucose concentration (8.3-33.4 mM) in the perfusion medium. Mean f SEM; 3-8 experiments in each group. l lp < compared with con0.01, **+p < 0.001,
8.3
trols (0).
16.7 25.0 glucose concenlrotion
33.4
(m&l)
(16.7 mM) insulin secretion (p < 0.05) and decreased plasma cholesterol levels over a wide range of doses tested (Figs. 3 and 4) (p < 0.001). Tj (Fig. 3) was five times more potent than T4 (Fig. 4). There was a sigmoidal relationship between insulin secretion from the perfused pancreas and glucose concentration in the perfusion medium (Figs. 5 and 6). T3 (200 pg/kg/day for 5 days) and T4 (1000 pg/kg/day, for 5 days) had the same hyperthyroid potency, as shown by their inhibitory effect on plasma cholesterol (Table 1). Both TS and T., had identical inhibitory effects on insulin secretion from the pancreas in relation to glucose concentration in the perfusion medium (Figs. 5 and 6). In the presence of 8.3 mM glucose in the perfusion medium insulin secretion was not inhibited because 60-min cumulative insulin secretion values (Figs. 5 and 6) represent mainly the inhibition of
62 ‘C 5. E 0 e49 L3!i
*
$2.c, 1.
3
20.
0
8.3
16.7 25.0 glucose concentration
33.4
(mM)
Fig. 6. Effect of T4 (1000 &kg/day for 5 days) (e) in comparison with controls (0) on insulin secretion from the rat pancreas in relation to glucose concentration (8.3-33.4 mm) in the perfusion medium. Mean f SEM; 4-8 experiments in each group. lp < 0.05; l**p < 0.001 compared with controls (0).
86
SIGURD
Table 1. Effect of T3 (200 &kg/day Equivalent
Volume of Saline (Control)
Treatment
for 5 days), T, (1000 on Plasma Cholesterol ”
pg/kg/day
for 5 days), or an
levels in Rats (Mean f
19
54.9
T3
12
41 .o * 3.9*
16
41.2
TA
SEM)
Plasma Cholesterol (mg/lOO ml)
Control
*p < 0.01 compared
LENZEN
f
2.5
* 3.3*
with control.
the late phase of glucose-induced insulin secretion,’ and the late phase of secretion is not yet present at such a low glucose concentration.4 At higher glucose concentrations (16.7,25.0, and 33.4 mM) in the perfusion medium insulin secretion was significantly inhibited after treatment of the rats with Tj as well as T4 due to their effect on the late phase of secretion. Figures 5 and 6 indicate that the K, of the sigmoidal concentration-response curve was not affected, whereas V,,,,, was decreased (Figs. 5 and 6). DISCUSSION
The inhibitory effect of thyroid hormones on insulin secretion can be characterized as follows: (1) The speed of onset of the inhibitory effect of Tj is more rapid and the time required to develop its maximal inhibitory effect is shorter, whereas the duration of the inhibitory effect of T4 is longer and weeks are needed for complete regression of the inhibitory effect of T4 (Figs. 1 and 2). (2) The inhibitory effects of TJ and T4 are dose dependent (Figs. 3 and 4). (3) TJ is five times more potent than T4 (Figs. 3-6). The effects of T3 as well as T4 on plasma cholesterol levels as a parameter of the hyperthyroid effect correlate with those on insulin secretion, supporting the conclusion drawn from earlier experiments’ that inhibition of glucoseinduced insulin secretion is a specific effect of thyroid hormones. None of the different experimental situations provided evidence for a stimulatory effect of thyroid hormones on glucose-induced insulin secretion, though this has been often supposed (for discussion, see Lenzen et al.‘). At present I can provide no explanation for the more rapid inhibitory effect of thyroid hormones on plasma cholesterol levels, the only discrepancy between the effect of thyroid hormones on cholesterol levels and insulin secretion. The different pharmacokinetic characteristics and the generally known greater biologic potency of Tj in comparison to T4 are unexplained phenomena.” Two alternatives are being considered at present and both have to be considered as possible explanations: ” (I) T4 may be a prohormone that must be converted to Tj to exert hormonal effects; or (2) the great differences in T3- and T,-binding properties on either side of the cell membrane may explain these findings. The fact that thyroid hormones that are diabetogenic in animals and in man’ can induce a state of hyperthyroidism in animals as well as in man makes it possible to draw conclusions that may help to improve the understanding of diabetes in man. Such conclusions cannot be drawn from studies on animals with genetic or chemical diabetes because such states cannot be reproduced in man. The present dose-response studies (Figs. l-6) together with results obtained
TH AND INSULIN
a7
SECRETION
in earlier studies2*4*5allow the characterization of experimental thyroid diabetes in animals as an experimental diabetes model. All the features are also known to be typical characteristics of human nonobese maturity-onset diabetes:‘**‘3 (1) selective and dose-dependent inhibition of glucose-induced insulin secretion of tolbutamide-, glyceraldehyde-, and (Figs. 3 and 4); 4~‘2(2) no inhibition mannose-induced insulin secretion;4’5”2 (3) absence of an immediate insulin secretory response of the pancreas from thyroid hormone-treated rats in response to a low glucose stimulus (8.3 mM) in contrast to a significant response in controls, as described in the results section’2*‘3 (the reason for this phenomenon is apparently the fact that an increase in the glucose concentration of the perfusion medium from 0 to 8.3 mM glucose was not sufficient to induce an immediate insulin secretory response of the pancreas from rats treated with T3 or T4 because these animals had increased plasma glucose levels in vivo already.2*4 In the presence of higher glucose concentrations (16.7-33.4 mM) in the perfusion medium the immediate insulin secretory response of the pancreas was not inhibited by T3 or T4 treatment of the rats); (4) selective inhibition of the late phase of glucose-induced insulin secretion (Figs. 3 and 4);4*5S’2*‘3 (5) reduction of the inhibition of glucose-induced (16.7 mM) insulin secretion after preperfusion of the pancreas with a substimulatory glucose concentration (5.5 mM) (unpublished observation);‘* (6) reduction of the relative inhibition of insulin secretion in the presence of a high stimulatory glucose concentration (33.4 mM) (Figs. 5 and 6);5 and (7) reduction of the relative inhibition of insulin secretion after feeding of the rats before the experiments.5.‘2 Two conclusions can be drawn from the experiments: (1) Inhibition of glucose-induced insulin secretion from the perfused pancreas is a specific effect of thyroid hormones characterized by features that are also known to be typical for nonobese maturity-onset diabetes in man. (2) Experimental hyperthyroidism in animals and hyperthyroidism in man have similar characteristics.‘** These similarities make it possible to estimate the relevance of results obtained in animal studies on thyroid diabetes for the understanding of diabetes in man. ACKNOWLEDGMENT The skillful technical
assistance
of S. Detels and R. LotTIer is gratefully
acknowledged.
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9. Grodsky GM: A threshold distribution hypothesis for packet storage of insulin and its mathematical modeling. J Clin Invest 51:2047, 1972 IO. Pittman CS, Pittman JA: Relation of chemical structure to the action and metabolism of thyroactive substances, in Greer MA, Solomon DH (eds): Handbook of Physiology. Section 7: Endocrinology, vol 3, Thyroid. Washington, DC, American Physiological Society, 1974, pp 233-253 1I. DiStefano JJ, Fisher DA: Peripheral
SIGURD LENZEN
distribution of the thyroid hormones. A priPharmacol marily quantitative assessment. Ther [B] 2:539, I976 12. Cerasi E: Insulin secretion: Mechanism of the stimulation by glucose. Rev Biophys 8:l. 1975 13. Schulz B, Michaelis D, Neumann 1. Gottschling D, Bibergeil H: Kohlenhydrattoleranz und Insulinverhalten im Glukoseinfusionstest bei normgewichtigen Personen mit Protodiabetesverdacht. Z Med 5: 138, 1976
