- Email: [email protected]
Reduced sensitivity to pirenzepine-induced blockade of growth hormone responses to arginine, exercise, and growth hormone-releasing hormone in type I diabetic subjects
Reduced Sensitivity to Pirenzepine-Induced Blockade of Growth Hormone Responses to Arginine, Exercise, and Growth Hormone-Releasing Hormone in Type I Diabetic Subjects V. Coiro,
M. Passeri,
E. Gardini,
L. Capretti,
L. Bianconi, The present
study
hormone
(GH)
inhibitory
effect
g infused
was performed
release
intravenously
GH-releasing exercise
(diabetics,
subject
administered. exercise
arginine four
controls.
of the was
experiments
with
I diabetic
pirenzepine. GH release GHRH
Again,
mechanisms
toward GHRH
lower
in the diabetic
than These the
Results with data
activity
responsible
for the hyperresponsiveness
by W.B.
at hypothalamic
Saunders
men, show
responses
cholinergic Q 1990
patients
and/or
in the
than
whereas
to arginine
from
complete
exercise,
level
might
and exercise
normal
to and explain in type
and with
when Since toward
in additional in controls
almost
lower
completely
30
(30
25,
in type
this
phenomenon
test
groups
Each
or exercise
might agent,
of normal
was
(N =
reflect further 7) and
in the absence
of the the
receptor
of
GHRH-induced GH response
blockade
patients.
and might
and 30 mg
and by 20 mg in the
and diabetics abolished
to
a dose-related
results
inhibition
failed
4). and to
pirenzepine
the blocking
I diabetic
or l-44
7) tests.
arginine mg
these
cholinergic
GHRH
I diabetic
20,
20 to 30 mg produced
controls.
muscarinic
the
infusion
(N =
N =
with
in the arginine
a significantly
growth
fashion
mg pirenzepine subjects
7: normals,
to stimulation
to GHRH
produced sensitivity
N =
almost
to arginine
of 17.5
without
ranging
performed
30 mg pirenzepine
arginine,
pituitary
tested
(diabetics,
influences
of 75 W for 30 minutes),
4) and normal
hyporesponsiveness
GH responses
An
to
in the
increased
be, at least
in part,
patients.
Company.
A
N ABNORMAL regulation of growth hormone (GH) secretion affects patients with type I diabetes mellitus, who often show elevated diurnal and sleep-related GH secretion’.” and exaggerated GH responses to physical exercise,‘,’ and pharmacological stimulation with arginine infusion.’ Muscarinic cholinergic receptors have been found in the mechanisms underlying the GH response to these stimulations,’ and thus alterations in the cholinergic control of GH secretion in type 1 diabetes mellitus might be supposed. Recent studies found the cholinergic receptor blocker pirenzepine capable of suppressing the GH response to various releasing stimuli in diabetics, as well as in normal subjects.‘.” These studies were performed with high amounts of pirenzepine and established that, as in normal subjects, GH secretion in diabetics was modulated by pirenzepinesensitive cholinergic pathways. However, results could not clarify whether diabetics had an altered sensitivity to muscarinic receptor blockade. The purpose of the present study was to evaluate in a From the University Clinics of Internal Medicine, Endocrinology, School of Medicine, University of Parma, Parma; Division of Cardiology, Hospital of Parma, Parma; Division of Internal Medicine, Hospital of Codogno, Codogno; Division of Internal Medicine, Hospital of Cremona. Cremona, Italy. Address reprint requests to Vittorio Coiro. MD, Cattedra di Clinica Medica Generale, Universitb di Parma. via Gram& 14. 43100 Parma. Italy. @ 1990 by W.B. Saunders Company. 0026-0495/90/3907-0002$03.00/O 668
was
or 30 mgj were similar
a decreased to
at doses
than
rather
20 or 25 mg pirenzepine
in normal GH
(0.20.25,
were
GH responses
which
system
in a dose-response
IV injection (N =
by 20 or 25 mg pirenzepine
for GH release showed
study,
subjects
administered
produced
F. Fagnoni,
the cholinergic
test at an intensity
71 or exercise higher
exercise,
Maffei,
on the GH responses
in both diabetic
other N =
M.L.
investigated
pirenzepine
In a preliminary
presented
and
inhibition
plus pirenzepine
treatment
underlying
patients
we
ergometer
infusion
7; normals,
pirenzepine
the stimuli
7) subjects.
groups.
to arginine
arginine
the percent
in diabetics
in both
to
significantly
(N =
in an IV bolus).
N =
Diabetic
response
hyperresponsiveness type
times.
(bicycle
by which
Therefore,
antagonist
N = 4). Therefore,
(diabetics,
In both groups,
GH
However,
test
(1 wg/kg
C. Davoli,
and P. Chiodera
the mechanism patients.
receptor exercise
in the GH response
tested
normal
inhibition
(GHRH)
whether
I diabetic
cholinergic
N = 4; normals,
during
was
in type
(IV) in 30 minutes),
hormone
modification
pirenzepine
to establish
altered
of the muscarinic
produce
than
was
G. Speroni,
R. Volpi,
dose-response fashion the effect of pirenzepine on the arginineor exercise-induced GH release in diabetic and normal subjects. Furthermore, since GH-releasing hormone (GHRH) is known to produce normal GH responses in diabetic subjects.“.‘” additional experiments with GHRH plus increasing amounts of pirenzepine were performed to test the effect of muscarinic blockade in normal and diabetic subjects, when GH is released in response to a comparable stimulus, MATERIALS
AND
METHODS
Subjects with similar body constitution were selected to participate in this study. Twenty-nine insulin-dependent diabetic men (mean weight 2 SE, 69.2 + 6.1 kg; height, 1.72 t 0.1 m; mean body mass index [BMI], 23.4; mean age, 31.9 2 2.2 years, and range, 24 to 44) gave their informed consent. Duration of diabetes ranged from 5 to 25 years (mean, 16.0). From the onset of their disease, the patients had been treated with insulin and at the timeof this study all of them were hospitalized for adjustment of insulin therapy in order to achieve improved metabolic control. After adjustment of insulin therapy, patients were discharged from the hospital and were observed by a day hospital. Their metabolic status was carefully monitored during the month following hospitalization, with three daily determinations of blood glucose, twice a week. Insulin therapy did not need to be changed in any of the patients; the metabolic status remained constant in all diabetics. The tests were performed during this period. None of the patients had clinical features or laboratory evidence of ketosis or any signs of associated endocrine. renal, hepatic, or other intercurrent disease. Subjects with no signs of retinopathy were chosen to participate in this study. Experiments were started after adjustment of insulin therapy (dose. 24 to 50 U/d; mean, 36 U/d of the intermediate duration monocomponent insulin Metabolism, Vol 39, No 7 (July), 1990: on 668-675
CHOLINERGIC CONTROL OF GH IN DIABETICS
plus short-acting
monocomponent
insulin,
given together
669
once or
twice daily), which remained unchanged during the period of this investigation. The last injection of insulin before each test was given on the day preceding each experimental day. As indices of control of the metabolic status of these patients, blood levels of glucose were measured at 7:00 AM, I 1:OO AM, and 5:OO PM on the days preceding the tests; for each patient. a mean value for blood glucose was obtained by averaging these three determinations. On the experimental days blood samples also were taken for measurement of glycosylated hemoglobin (HbA,,). Furthermore. 24-hour urinary glucose excretion was collected at the time of the tests. Twenty-nine normal men (mean weight * SE, 71.4 + 7.2 kg; height. 1.71 i 0.08 m; BMI, 24.5; mean age, 30.3 t 1.3 years, and range, 24 to 41) without any signs of endocrine disease or family history of diabetes mellitus were studied as controls. Normal and diabetic men were tested with exercise, arginine, or GHRH in the presence or absence of pirenzepine. Preliminary
Tests
Eight normal men and eight diabetics were tested with 15.0 and 17.5 mg pirenzepine during the arginine test (four normal men and four diabetics) and the exercise test (the remaining eight subjects), in order to determine the minimal effective dose of the drug capable of modifying the GH response to arginine or exercise stimulation. In the control tests, normal saline (NaCI 0.9%) was administered instead of pirenzepine. Experiments were performed as described below. Since neither 15.0 nor 17.5 mg pirenzepine modified the GH response during arginine and exercise tests (data not presented), we decided to use higher doses of pirenzepine in the following tests. Other subjects participated in these tests in order to avoid an excessive number of experiments in the same subjects. Exercise
Test
Seven normal controls and seven diabetics were tested with exercise. Exercise tests were performed on an bicycle ergometer at an intensity of 75 W from 0 to 30 minutes. Experiments started at 8:00 AM after overnight fast. Exercise started 15 minutes after the insertion of an intravenous (IV) catheter into an antecubital vein, which was used for blood sampling. Basal blood samples were taken at time 0 (just before exercise). Further specimens were withdrawn at time 10, 20, 30. 40, 50, and 60 minutes. When the effect of pirenzepine on the GH response to exercise was tested, an equal procedure was followed, except for the injection of 20, 25, or 30 mg pirenzepine at time - 10 minutes. In the control test, an equal amount of normal saline was injected instead of pirenzepine. Control and experimental tests were performed in random order, with an interval of at least 5 days. .4rginr’ne
Test
Seven normal controls and seven diabetics were tested with arginine. At 8:00 AM of the experimental day, two indwelling catheters were inserted into forearm veins of opposite arms of subjects fasting from the previous evening and lying in the recumbent position. One catheter was used for blood sampling, the other for arginine infusion. In one test (control test), a blood specimen (0 minutes) was withdrawn before the beginning of arginine (arginine monoh,ydrochIoride, 30 g administered IV over a 30-minute period) infusion and further samples were taken at time 15, 30, 45, 60, and 90 minutes. In the other tests (experimental tests), a similar protocol was followed, except that 20,25, or 30 mg pirenzepine (Gastrozepin, Boehringer Ingelheim, Italy) was administered IV 10 minutes before arginine infusion. In the control test, an equal amount of normal saline was injected instead of pirenzepine. Control and experimental tests were performed in random order. with an interval of at least 5
days. In all subjects.
blood samples
were used for measurements
of
glucose, GH, and glucagon concentrations. Somatomedin-C (SMC), free fatty acids (FFA), and HbA,, concentrations were evaluated in the specimen taken at time 0 of all tests. GHRH
Test
Seven normal controls and seven diabetics were tested with GHRH. Experiments were performed as described for the arginine test, except that 1 fig/kg body weight GHRH (l-44, Sanofi, Toulouse, France) was given at time 0 in an IV bolus instead of arginine. All blood specimens were used for measurements of glucose and GH concentrations. SM-C. FFA, and HbAlC levels were measured in the sample taken at time 0 of all tests. During exercise, subjects breathed through a low-resistence, one-way valve connected to a P.K. Morgan measurement system (Avinton, Seattle, WA), which had been appropriately calibrated. The following parameters were measured: ventilation, frequency of breathing, tidal volume, oxygen consumption (VO?), carbon dioxide production (VCO?), and respiratory exchange ratio (R). Determination of heart rate and blood pressure were carried out by an experienced cardiologist. Heart rate was measured by auscultation over the precordium; blood pressure was evaluated with a sphygmomanometer. Assays All samples from a single subject were determined in duplicate in the same assay. GH concentrations in the serum were measured in all samples by a specific radioimmunoassay (RIA),lh using a double-antibody technique. Intraassay and interassay coefficients of variation were 3.6% and 8%. respectively. The lower limit of sensitivity was 0.5 ng/mL. Glucagon was measured by specific RIA,” using a double-antibody technique. The intraassay and interassay coefficients of variation were 5% and 6.3%. respectively. The detection limit was 14.5 pg/mL. SM-C levels were measured by RIA (double-antibody method) using kits obtained by Nichols Institute Diagnostics (San Juan Capistrano. CA), as described previously.‘x The intraassay coefficient of variation was 5% and the interassay coefficient of variation was 10%. The sensitivity of the assay was 0.1 mU/mL. FFA concentrations were determined using an enzyme-based method.19 HbA,, was assayed by high-pressure liquid chromatography after hemolysis of the red blood cells,‘O~” using reagents obtained from BIO-Rad Laboratories (Richmond, CA) (normal range. 4.15% to 9.08%). Blood glucose was measured with an IL 918 autoanalyzer (Instrumentation Laboratory, Milan, Italy), using a glucose oxidase-peroxidase procedure. Statistical analyses were performed using the Kruskall-Wallis test, analysis of variance randomized factorial block (RBF [two-way ANOVA]), ANOVA split-splot factorial design, and the Sperman’s r linear coefficient correlation, as appropriate. The areas under the curve of the glucagon and GH responses to arginine were calculated with trapezoid integration. Data are reported as mean -r SE. RESULTS
The
indices
diabetic Arginine Blood test
of control
patients
the
metabolic
in Table
status
in the
1.
Test glucose
were
levels
184.4
(time
+ I5
218.4
k 10.4 (time
minutes), values
of
are reported
minutes), and
were
+
219.4
observed
of diabetic 9.8
208.7 +45 2
patients
mg/dL k
(time 10.0
minutes), 12.1
during
(time arginine
during 0).
(time
223.8 +90
arginine
198.3 +30
+
8.8
minutes),
+ 11.6 (time minutes).
plus pirenzepine
+60
Similar tests.
COIRO ET AL
670
Table 1. Clinical and Biochemical Characteristics
Age Iv) Arginine test group (n = 7) Mean + SE 33.7 Range
i- 2.0
Range
Range
35.6
12-25
+ 2.7
f 1.7
k 3.0
35.0
lo-23
188.3
-r 2.5
38.7
5-19
HbA,,
+ 9.9’
9.0 ? 0.8’
168.4-193.5
182.0
24-50
15.1 t 1.6
24-37
AverageBlood GlucoseIfig/DLl
23-40
16.3 + 1.8
25-44
Exercise test group (n = 7) Mean + SE 28.8
InsulinDose W/24 h)
16.7 + 2.7
26-42
GHRH test group (n = 71 Mean t SE 33.2
of the Three Groups of Diabetic Patients
Diabetes Duration(yr)
8.0-9.4
k 6.3*
8.9 i 0.4’
158.4-197.4
+ 2.3
186.2
29-48
7.9-9.6
f 10.2’
9.2 i 0.7’
160.6-195.0
8.1-9.5
‘Mean of 4 test days at time 0.
30 mg pirenzepine in both groups. When 25 mg pirenzepine was administered, the inhibition observed in the control subjects (85% decrease of the mean peak response) was still significantly higher (P < .Ol) than in the diabetic patients (60%) (Fig 2). The incretory response was significantly lower in the controls than in the diabetic patients (F = 16.710. P < .OOl) (Fig 1). In both groups, the GH response to arginine was almost completely blocked with the administration of 30 mg pirenzepine (Figs 1 and 2). The basal levels of glucagon were similar in normal and diabetic subjects (Table
The basal concentration (mean of 4 test days) of SM-C (diabetics, 0.7 + 0.06 mU/mL; normals, 0.8 + 0.05) and FFA (diabetics, 0.81 k 0.09 mmol/L; normals, 0.72 f 0.08) were similar in the two groups. The results of the arginine test performed without pirenzepine and after treatment with different amount of the cholinergic antagonist are shown in Figs 1 and 2. The basal concentrations of GH were similar in the normal controls (1.5 + 0.1 ng/mL) and diabetic patients (1.8 k 0.2 ng/mL). Serum GH levels were significantly increased by arginine infusion in both groups. The mean peak concentration of GH was reached at 45 minutes in both control and diabetic subjects (P < .OOl v time 0); however, the incretory response was significantly higher in the diabetic patients than in the control group (F = 10.316, P < .002). Correlation analyses of the peak GH response to arginine in diabetics with basal and 45-minute blood glucose levels or basal HbAlC, FFA, and SM-C did not provide significant results. Administration of 20 mg pirenzepine strikingly decreased the mean peak GH response by 70% in the normal subjects, but only by 30% in the diabetic patients (P < .Ol) (Fig 2). The incretory response was significantly lower in the control group than in the diabetic patients (F = 37.906, P < .OOl) (Fig 1). Higher inhibitory effects were obtained with 25 and
2). The incretory response of glucagon was significantly higher in the diabetic patients than in the control group (F = 9.786, P -C .002) (Table 2). When GH and glucagon data of diabetic patients were considered together, correlation analyses of hormonal peaks or areas in response to arginine did not provide significant results. Administration of 20, 25 or 30 mg pirenzepine did not change the glucagon response to arginine in both normal and diabetic subjects (Table 2). Exercise Test Blood glucose levels in diabetic patients during exercise were 180.1 * 10.8 mg/DL (time 0), 188.4 + 9.7 (time + 10
Normals
0
IS
Diabetics
30
45
60
90
0
1s
JO
45
60
90
Minutes Fig 1. Serum GH response to arginine (A) alone or together with different doses of pirenrepine (P) in normal and diabetic subjects. Each point represents the mean t SE of seven observations. 0. A; +. A + P. 20 mg: n . A + P, 25 mg: 0, A + P, 30 mg.
CHOLINERGIC
CONTROL
OF GH IN DIABETICS
671
0
patients (30%) (P < .Ol) (Fig 4). The GH response to exercise was significantly lower in normal than in diabetic subjects (F = 25.265, P < .OOl) (Fig 3). Higher inhibitory effects were obtained with 25 and 30 mg pirenzepine in both groups. When exercise was performed after 25 mg pirenzepine, the GH response to exercise was significantly lower in the control than in the diabetic group (F = 9.985, P < ,002) (Fig 3). This amount of pirenzepine induced comparable percent inhibitions in diabetic and control subjects (Fig 4). In the exercise plus 30-mg pirenzepine test, GH responses to exercise (Fig 3) and percent inhibitions induced by pirenzepine were similar in diabetics and controls (Fig 4). Heart rate (diabetics: basal 75 e 8 bpm, peak 126 + 9; normals: basal 72 k 8, peak 123 t 8), mean blood pressure (diabetics: basal 97 * 5 mm Hg, peak 113 2 8; normals:basal 95 t 4, peak 109 + 7), respiratory rate diabetics: basal 12.6 k I .4 min ‘, peak 21.5 i 2.6; normals:basal 12.4 + 1.0. peak 20.3 t 2.0), ventilation (diabetics: basal 10.6 + 0.7 L/min, peak61.8 k 2.9; normals: basal 10.4 t 0.9, peak60.0 z 3.0), tidal volume (diabetics: basal 0.6 + 0.2 L. peak 2.0 k 0.4; normals: basal 0.8 IT 0.2, peak 1.8 +- 0.4). VO, (diabetics: basal 333 f 13. mL/min, peak 2,239 * 147; normals: basal 335 k 12, peak 2,234 f 150). VCO, (diabetics: basal 285 + 17 mL/min, peak 2,098 f 158; normals: basal 286 + 17, peak 2,070 + 151), R (diabetics: basal 0.86, peak 0.92; normals: basal 0.89, peak 0.93) were not significantly different between diabetic and normal subjects. Similar values were observed in both groups after treatment with 20, 25, and 30 mg pirenzepine.
-20
3 Q
-10
A.
-60 #
-80
yco.0 I
I
n.s. I
A
A+P20mg
A+P25mg
A+P30mg
Fig 2. Percentage decrease of the peak GH response (% LI GH. after subtraction of basal values) to arginine (A) induced by different amounts of pirenzepine (PI in normal (WI and diabetic (0) subjects. Each bar represents the mean + SE of seven observations.
minutes), 197.8 i 10.9 (time +20 minutes), 204.5 + 13.0 (time -t30 minutes), 215.0 k 12.4 (time +40 minutes), 221.1 :t 10.4(time +50minutes),and226.2 + 9.9 (time +60 minutes). Similar values were observed during exercise plus pirenzepine tests. The basal levels (mean of 4 test days) of SM-C (diabetics, 0.8 k 0.07 mU/mL; normals. 0.9 + 0.06) and FFA (diabetics, 0.85 * 0.10 mmol/L. normals, 0.68 2 0.07) were similar in the two groups. Basal GH levels were similar in diabetic (1.7 + 0.3 ng/mL) and normal (1.6 + 0.1 ng/mL) subjects. In both groups, exercise induced a significant increase in serum GH concentrations, with a peak response at 30 minutes (P < .OOl v time 0). The increment was significantly higher in the diabetic than in the normal subjects (F = 9.700, P < ,002) (Fig 3). The GH peak responses in diabetics were not statistically correlated with basal and 30-minute glucose concentrations and basal HbA,,, FFA, and SM-C levels. The administration of 20 mg pirenzepine significantly reduced the exercise-induced GH increase (Fig 3). However, a more marked inhibition was observed in the normal controls (60%) than in the diabetic Table 2. Glucagon Response to Arginine Administered
GHRH
Test
Blood glucose levels in diabetic patients during GHRH testing were 177.7 t 10.0 mg/dL (time 0). 178.0 i 9.8 (time + 15 minutes), 197.3 * 8.9 (time + 30 minutes), 206.0 f 7.8 (time +45 minutes), 215.9 + 8.4 (time +60 minutes), 222.9 + 10.6 (time +90 minutes), and 235.5 + 11.8 (time + 120 minutes). Similar values were observed during GHRH plus pirenzepine tests. The basal levels (mean of 4 test days) of SM-C (diabetics, 0.8 * 0.08 mU,‘mL; normals, 0.9 f 0.05) and FFA (diabetics, 0.84 +_0.09 mmol/L; normals, 0.70 k 0.09) were similar in the two groups. Basal GH levels were similar in normal (1.6 * 0.4 ng/mL) and diabetic
Alone or After Treatment
With Increasing Amounts of Pirenrepine
Glucagon(pg/mL) Treatment
Subjects
0 min
+15min
+30
+45
min
min
+60
mu”
f90
rn,”
Normals 94.7
+ 6.9
194.3
t
10.4
227.7
+ 19.4
184.7
+ 10.0
95.1
z 6.7
93.7
? 6.5
95.0
* 7.1
197.6
+ 11.8
225.4
+ 20.3
182.9
+ 11.4
93.7
i 6.0
92.8
+ 6.1
AfP25mg
93.0
‘- 5.9
200.1
f
12.0
229.5
c 19.8
180.4
+ 11.8
94.8
t 6.6
92.0
* 7.0
A+P
96.8
t 8.0
193.1
f
11.9
224.3
+ 19.7
178.8
i
12.0
95.8
t 6.8
91.9
2 8.1
A
AfP
20 mg 30 mg
Diabetics 104.0
? 6.7
245.4
+ 11.3’
293.7
? 18.0*
233.4
+ 11.8’
108.7
i- 8.9*
102.9
+ 8.2
A+P
20 mg
106.6
* 7.4
250.7
t
12.4’
296.4
+ 19.8*
240.6
? 12.0*
111.4
+ 9.7.
101.4
+ 8.7
A+P
25 mg
108.2
? 8.0
242.7
t
12.7t
289.7
+ 20.3*
230.9
t
13.0’
107.6
i 9.9t
102.5
f 7.9
A+P
30 mg
103.7
* 7.5
244.9
+ 12.0’
288.6
i 21.0*
233.6
+ 12.8”
106.5
t 9.5t
100.4
+ 8.4
A
NOTE.
Each point represents
Abbreviations: lP < .02,
A, arginine;
the mean
* SE of the observations.
P, pirenzepine.
tP < .05 between
normals
and diabetics
(Kruskall-Wallis
test).
COIRO ET AL
672
Normals
Diabetics
1
0
IO
20
30
40
50
60
0
IO
20
30
40
50
60
Mlrlutes Fig 3. Serum GH response to exercise (El alone or together with different doses of pirenzepine (P) in normal and diabetic subjects. Each point represents the mean + SE of seven observations. 0, E; +, E + P, 20 mg: n , E + P, 25 mg, 0, E + P, 30 mg.
(1.9 + 0.4) subjects. In both groups, GHRH induced a significant increase in serum GH concentrations, with a peak response at 15 minutes (P < ,001 v time 0). GH responses in the two groups were similar (Fig 5). The administration of 20 mg pirenzepine significantly reduced the GHRH-induced GH increase (Fig 5). However, normal controls showed a more marked inhibition (50%) than diabetic patients (22%) (P < .02) (Fig 6). The GH response to GHRH was significantly lower in normal controls than in diabetics (F = 29.899, P < ,001) (Fig 5). When 25 mg pirenzepine was administered, the inhibition obtained in the control subjects (70% decrease of the mean peak response) was significantly higher than in the diabetic patients (45%) (P < .02) (Fig 6). Thirty milligrams pirenzepine produced a similar and almost complete blockade of the GH response to GHRH in both groups (Figs 5 and 6). At all doses, pirenzepine induced a similar transient impairment of visual accommodation in all subjects. 0
-20 %
-40
bQ
-60
4
-80 11.5. -100
!
E
Et P20mg
E+P25mg
E * P3Omg
Fig 4. Percent decrease of the peak GH response 1% A GH. after subtraction of basal values) to exercise (E) induced by different amounts of pirenzepine (P) in normal (W) and diabetic (ml subjects. Each bar represents the mean + SE of seven observations.
DISCUSSION
The results of the present study confirm that in patients with type I diabetes mellitus there is a higher GH responsiveness to arginine infusion and physical exercise than in normal subjects. Abnormal GH responses in diabetes mellitus have been attributed to poor metabolic control.‘.“.” Our data do not support this hypothesis, since no correlations were found between peak GH responses to exercise or arginine and the metabolic status of our patients (blood glucose, FFA, and HbA,, levels). In addition, SM-C levels were not correlated with peak GH responses. In agreement with previous reports,*‘-” diabetic patients presented a higher glucagon increase than normal controls during arginine infusion. Glucagon is a well-known stimulator of GH release.‘7”9 and thus, the higher glucagon increase in diabetics might have induced the increased GH response to arginine. This possibility is unlikely, because no correlation was observed between glucagon and GH responses to arginine. However, if glucagon were responsible for the higher GH response to arginine in diabetics, it should have exerted this effect through muscarinic cholinergic mediation. In fact, in the presence of pirenzepine, the GH response to arginine was blunted, even though the glucagon increase remained unmodified. In both control and diabetic subjects, pirenzepine administered at concentrations ranging from 20 to 30 mg produced a dose-related decrease of the GH responses to stimulation with exercise or arginine. The inhibitory effect of pirenzepine was significantly smaller in diabetic than in normal subjects when lower doses of the drug were administered (20 and 25 mg in the arginine test and 20 mg in the exercise test), but it was similar in both groups when a saturating amount of the cholinergic antagonist (30 mg) was administered. The higher dose of pirenzepine necessary for suppression in diabetics might be interpreted as an effect of the altered hormonal milieu conditioning hyperresponsiveness to arginine and exercise in these subjects; for example, it might be attributed to the simultaneous higher glucagon response during argin-
CHOLINERGIC
CONTROL
OF GH IN DIABETICS
673
Normals l-
Diabetics
pa
,-
I I-
\i
\ I
0
15
30
I
45
I
1
60
90
1
0
120
15
30
45
60
90
120
Minutes Fig 5.. Serum GH response to GHRH alone or together with different doses of pirenzepine (PI in normal and diabetic subjects. Each point represents the mean + SE of seven observations. 0, GHRH; +, GHRH + P, 20 mg; n , GHRH + P, 25 mg, 0, GHRH + P, 30 mg.
ine infusion. This hypothesis was not confirmed by our additional tests with GHRH. In fact, even though GHRH induced similar GH responses in normal and diabetic subjects, the differences in pirenzepine action between groups were similar to those observed in the arginine and exercise tests. Taken together, these results suggest the existence of intrinsic changes in the cholinergic system of type I diabetics. The shift of the dose-response curve in the diabetic subjects indicates a lower sensitivity of the cholinergic system to muscarinic receptor blockade. However, we were unable to detect a change in diabetics of the minimal concentration of pirenzepine capable of inducing a statistically significant inhibition (20 mg in both control and diabetic subjects). The possible existence of different mini0
-40 3 -60
cl Be
-80
n.s
-100
GHRH
GHRH+ P 20mg
GHRH+ P 25mg
GHRH+ P 30mg
Fig 6. Percent decrease of the peak GH response (% A GH, after subtraction of basal values) to GHRH induced by different amounts of pirenzepine (PI in normal (ml and diabetic (al subjects. Each bar represents the mean * SE of seven observations.
ma1 effective doses in the range between IS and 20 mg pirenzepine might be masked by the poor sensitivity of our in vivo model. In light of our present results, the higher GH responsiveness of the diabetic patients to arginine and exercise might be attributed to an increased activity of cholinergic neurons. This phenomenon might be an aspect of a more general disorder affecting the neuroendocrine control of GH secretion in type 1 diabetic subjects. Particularly, the GH responses to exercise and arginine are also regulated by adrenergic stimuli.7~‘0 and thus it is likely that both cholinergic and adrenergic systems work in concert to tonically influence exercise- and arginine-induced GH secretion. In view of the well-known adrenergic alterations affecting type I diabetics3’-j5 our results might reflect a modification in diabetics of the possible interplay between adrenergic and cholinergic stimuli. The reduced effect of pirenzepine in our diabetic patients might be explained by changes in cholinergic muscarinic receptor number or sensitivity. Cholinergic receptors are present both at hypothalamic and pituitary level.3h~3” As pirenzepine is unable to cross the blood brain barrier (BBB)39 and our patients were not affected by diabetic retinopathy and thus presumably had a normal BBB permeability, cholinergic alterations in diabetics might be supposed at the pituitary level or in hypothalamic structures outside the BBB (ie, the median eminence). At present, there is no evidence for a direct cholinergic modulation of GH secretion at the pituitary level. In fact, atropine administered in vivo has been found capable of blocking the GHRH-induced GH secretion in humans, whereas atropine added in vitro was unable to change the GH response to GHRH in bovine pituitary cell culture.4” Changes at the level of the median eminence might be supposed. It has been reported that acetylcholine inhibits the release of somatostatin from the hypothalamus in vitro
674
CON0
through a muscarinic mechanism,” and thus, pirenzepine might enhance endogenous somatostatin release, blocking the GH response to arginine, exercise, and GHRH. In light of this hypothesis, our results might indicate that in diabetes mellitus there is a cholinergic receptor alteration affecting the mechanism controlling hypothalamic somatostatin re-
lease. Further studies are needed to substantiate esis.
ET AL
this hypoth-
ACKNOWLEDGMENT
We thank Dr Claudio Nava (Sanofi Midy, Milan Italy) for generously supplying GHRH.
REFERENCES I, Johansen K. Hansen AP: High 24-hour level of serum growth hormone in juvenile diabetics. Br Med J 2:356-357, 1969
17. Henquin JC. Malvaux P, Lambert AE: Glucagon immunoassay using polyethylene glycol to precipitate antibody-bound hormone. Diabetologia 10:61-68, 1974
2. Molnar GD. Taylor WF, Langworthy A, et al: Diurnal growth hormone and glucose abnormalities in unstable diabetics: Studies of ambulatory fed subjects during continuous blood glucose analysis. J Clin Endocrinol Metab 34:837-846, 1972
18. Furlanetto RW, Underwood LE. Van Wyk JJ, et al: Estimation of somatomedin C levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest 60:648-657, 1977
3. Press M, Tamborlane WV, Sherwin RS: Importance of raised growth hormone levels in mediating the metabolic derangements of diabetes. N Engl J Med 31:810-815. 1984
19. Mizuni K. Toyosato M, Yabumoto method for calorimetric determination Biochem 108:6-IO, 1980
4. Vigneri R, Squatrito S, Pezzino V, et al: Growth hormone levels in diabetes: Correlation with the clinical control of the disease. Diabetes 25: 167- 172, 1976
20. Koenig RJ. Cerami A: Synthesis of hemoglobin A,, in normal and diabetic mice: Potential model of basement membrane thickening. Proc Nat1 Acad Sci 72:3687-3691, I975
5. Yde H: The immunoreactive growth-hormone in serum from patients with various types of diabetes mellitus. Acta Endocrinol 64~339-346. 1970
21. Koenig RJ, Araujo DC, Cerami A: Increased in diabetic mice. Diabetes 25: l-5, 1976
6. Hansen AP: Abnormal serum growth hormone response exercise in juvenile diabetics. J Clin Invest 49:1467-1478, 1970
to
S, et al: A new enzymatic of free fatty acid. Anal
hemoglobin
A,,
22. Hansen AP, Johansen K: Diurnal patterns of blood glucose. serum free fatty acids, insulin, glucagon and growth hormone in normals and juvenile diabetics. Diabetologia 6:27-33, 1970
7. Hansen AP: The effect of adrenergic receptor blockade on the exercise-induced serum growth hormone rise in normals and juvenile diabetics. J Clin Endocrinol Metab 33:807-812, 1971
23. Johansen K, Hansen AP: Diurnal serum growth levels in poorly and well-controlled juvenile diabetics. 20~239-245, 1971
8. Burday SZ, Fine PH. Schalch DS: Growth hormone secretion in response to arginine infusion in normal and diabetic subjects: Relationship to blood glucose levels. J Lab Clin Med 71:897-911, 1968
24. Aguilar-Parada E. Eisentraut AM, Unger RH: Pancreatic glucagon secretion in normal and diabetic subjects. Am J Med Sci 257:415-419, 1969
9. Arends J, Wagner ML, Willms BL: Cholinergic muscarinic receptor blockade suppress arginine- and exercise-induced growth hormone secretion in type I diabetic subjects. J Clin Endocrinol Metab 66:389-395, 1988 IO. Chiodera P. Coiro V. Speroni G. et al: The growth hormone response to thyrotropin-releasing hormone in insulin-dependent diabetics involves a cholinergic mechanism. J Clin Endocrinol Metab 59:794-797, 1984
I I. Delitala G, Tomasi P. Virdis R: Neuroendocrine human growth hormone secretion. Diagnostic tion. J Endocrinol Invest 11:441-464, 1988
regulation of and clinical applica-
12. Pietschmann P, Schernthaner G, Luger A: Effect of cholinergic muscarinic receptor blockade on human growth hormone (GH)-releasing hormone (I-44)-induced GH secretion in acromegaly and type I diabetes mellitus. J Clin Endocrinol Metab 63:389393. 1986 13. Giampietro 0, Ferdeghini M. Miccoli R, et al: Effect of growth hormone-releasing hormone and clonidine on growth hormone release in type 1 diabetic patients. Horm Met Res 19:636-641, 1987 14. Kopelman PG, Mason AC, Noonan K. et al: Growth hormone response to growth hormone releasing factor in diabetic men. Clin Endocrinol28:33-38, 1988 15. Press M, Tamborlane response to growth hormone 33:804-806, 1984
WV, Thorner MO, et al: Pituitary releasing factor in diabetes. Diabetes
16. Schalch DS, Parker ML: A sensitive double antibody immunoassay for human growth hormone in plasma. Nature 203:1141I142,1964
25. Aronoff SL. Bennet stimulated hyperglucagonemia 407, 1976
hormone Diabetes
PH, Rushforth NB, et al: Argininein diabetic subjects. Diabetes 25:404-
26. Unger RH, Aguilar-Parada E, Muller WA. et al: Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837-848, 1970 27. Mitchell ML, Byrne MJ. Silver J: Growth hormone release by glucagon. Lancet 1:289-290, 1969 28. Rao RH. Spathis GS: lntramuscolar glucagon as a provocative stimulus for the assessment of pituitary function: Growth hormone and cortisol responses. Metabolism 37:659-663. 1987 29. Wieland RG, Hallberg MC, Zorn EM: Growth hormone response to intramuscolar glucagon. J Clin Endocrinol Metab 37:329-330, 1973 30. Buckler JMH, Bold AM, Taberner M, et al: Modification of hormonal responses to arginine by cu-adrenergic blockade. Br Med J 3:153-154, 1969 31. Bolli G, Compagnucci P, Cartechini MG, et al: Urinary excretion and plasma levels of norepinephrine and epinephrine during diabetic ketoacidosis. Acta Diabetol Lat 16:157-167, 1979 32. Christensen NJ: Plasma norepinephrine and epinephrine in untreated diabetics, during fasting and after insulin administration. Diabetes 23: l-8. 1974 33. Robertson RP. Halter JB, Porte DJ Jr: A role for alphaadrenergic receptors in abnormal insulin secretion in diabetes mellitus. J Clin Invest 57:791-795, 1976 34. Speroni G, Ceda GP, Capretti L, et al: Clonidine and GH secretion in insulin dependent diabetes (IDD). Horm Metab Res 15146-47. 1983 35. Tamborlane
WV, Sherwin
RS, Koivisto V, et al: Normaliza-
CHOLINERGIC CONTROL OF GH IN DIABETICS
tion of the growth hormone and catecholamine response to in juvenile-onset diabetic subjects treated with a portable infusion pump. Diabetes 28:785-788. 1979 36. Burt DR. Taylor RL: Muscarinic receptor binding anterior pituitary. Neuroendocrinology 30:344-349. 1980 37. Schaeffer JM, Hsueh AJW: Acetylcholine in the rat pituitary gland. Endocrinology 106:1377-1381. 1980 38. Taylor RL, Burt DR: Pituitary cell cultures contain inic receptors. Em J Pharmacol 65:305-308, 1980
675
exercise insulin in sheep anterior muscar-
39. Hammer R, Koss FW: The pharmacokinetic profile of pirenzepine. Scan J Gastroenterol 14:l-20. 1979 (suppl57) 40. Casanueva FF. Villanueva L, Dieguez C, et al: Atropine blockade of growth hormone (GH)-releasing hormone-induced GH secretion in man is not exerted at pituitary level. .J Clin Endocrinol Metab62:186-191, 1986 41. Richardson SB, Hollander CS. D’Eletto R, et al: Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology 107: 122- 129. 1980
M. Passeri,
E. Gardini,
L. Capretti,
L. Bianconi, The present
study
hormone
(GH)
inhibitory
effect
g infused
was performed
release
intravenously
GH-releasing exercise
(diabetics,
subject
administered. exercise
arginine four
controls.
of the was
experiments
with
I diabetic
pirenzepine. GH release GHRH
Again,
mechanisms
toward GHRH
lower
in the diabetic
than These the
Results with data
activity
responsible
for the hyperresponsiveness
by W.B.
at hypothalamic
Saunders
men, show
responses
cholinergic Q 1990
patients
and/or
in the
than
whereas
to arginine
from
complete
exercise,
level
might
and exercise
normal
to and explain in type
and with
when Since toward
in additional in controls
almost
lower
completely
30
(30
25,
in type
this
phenomenon
test
groups
Each
or exercise
might agent,
of normal
was
(N =
reflect further 7) and
in the absence
of the the
receptor
of
GHRH-induced GH response
blockade
patients.
and might
and 30 mg
and by 20 mg in the
and diabetics abolished
to
a dose-related
results
inhibition
failed
4). and to
pirenzepine
the blocking
I diabetic
or l-44
7) tests.
arginine mg
these
cholinergic
GHRH
I diabetic
20,
20 to 30 mg produced
controls.
muscarinic
the
infusion
(N =
N =
with
in the arginine
a significantly
growth
fashion
mg pirenzepine subjects
7: normals,
to stimulation
to GHRH
produced sensitivity
N =
almost
to arginine
of 17.5
without
ranging
performed
30 mg pirenzepine
arginine,
pituitary
tested
(diabetics,
influences
of 75 W for 30 minutes),
4) and normal
hyporesponsiveness
GH responses
An
to
in the
increased
be, at least
in part,
patients.
Company.
A
N ABNORMAL regulation of growth hormone (GH) secretion affects patients with type I diabetes mellitus, who often show elevated diurnal and sleep-related GH secretion’.” and exaggerated GH responses to physical exercise,‘,’ and pharmacological stimulation with arginine infusion.’ Muscarinic cholinergic receptors have been found in the mechanisms underlying the GH response to these stimulations,’ and thus alterations in the cholinergic control of GH secretion in type 1 diabetes mellitus might be supposed. Recent studies found the cholinergic receptor blocker pirenzepine capable of suppressing the GH response to various releasing stimuli in diabetics, as well as in normal subjects.‘.” These studies were performed with high amounts of pirenzepine and established that, as in normal subjects, GH secretion in diabetics was modulated by pirenzepinesensitive cholinergic pathways. However, results could not clarify whether diabetics had an altered sensitivity to muscarinic receptor blockade. The purpose of the present study was to evaluate in a From the University Clinics of Internal Medicine, Endocrinology, School of Medicine, University of Parma, Parma; Division of Cardiology, Hospital of Parma, Parma; Division of Internal Medicine, Hospital of Codogno, Codogno; Division of Internal Medicine, Hospital of Cremona. Cremona, Italy. Address reprint requests to Vittorio Coiro. MD, Cattedra di Clinica Medica Generale, Universitb di Parma. via Gram& 14. 43100 Parma. Italy. @ 1990 by W.B. Saunders Company. 0026-0495/90/3907-0002$03.00/O 668
was
or 30 mgj were similar
a decreased to
at doses
than
rather
20 or 25 mg pirenzepine
in normal GH
(0.20.25,
were
GH responses
which
system
in a dose-response
IV injection (N =
by 20 or 25 mg pirenzepine
for GH release showed
study,
subjects
administered
produced
F. Fagnoni,
the cholinergic
test at an intensity
71 or exercise higher
exercise,
Maffei,
on the GH responses
in both diabetic
other N =
M.L.
investigated
pirenzepine
In a preliminary
presented
and
inhibition
plus pirenzepine
treatment
underlying
patients
we
ergometer
infusion
7; normals,
pirenzepine
the stimuli
7) subjects.
groups.
to arginine
arginine
the percent
in diabetics
in both
to
significantly
(N =
in an IV bolus).
N =
Diabetic
response
hyperresponsiveness type
times.
(bicycle
by which
Therefore,
antagonist
N = 4). Therefore,
(diabetics,
In both groups,
GH
However,
test
(1 wg/kg
C. Davoli,
and P. Chiodera
the mechanism patients.
receptor exercise
in the GH response
tested
normal
inhibition
(GHRH)
whether
I diabetic
cholinergic
N = 4; normals,
during
was
in type
(IV) in 30 minutes),
hormone
modification
pirenzepine
to establish
altered
of the muscarinic
produce
than
was
G. Speroni,
R. Volpi,
dose-response fashion the effect of pirenzepine on the arginineor exercise-induced GH release in diabetic and normal subjects. Furthermore, since GH-releasing hormone (GHRH) is known to produce normal GH responses in diabetic subjects.“.‘” additional experiments with GHRH plus increasing amounts of pirenzepine were performed to test the effect of muscarinic blockade in normal and diabetic subjects, when GH is released in response to a comparable stimulus, MATERIALS
AND
METHODS
Subjects with similar body constitution were selected to participate in this study. Twenty-nine insulin-dependent diabetic men (mean weight 2 SE, 69.2 + 6.1 kg; height, 1.72 t 0.1 m; mean body mass index [BMI], 23.4; mean age, 31.9 2 2.2 years, and range, 24 to 44) gave their informed consent. Duration of diabetes ranged from 5 to 25 years (mean, 16.0). From the onset of their disease, the patients had been treated with insulin and at the timeof this study all of them were hospitalized for adjustment of insulin therapy in order to achieve improved metabolic control. After adjustment of insulin therapy, patients were discharged from the hospital and were observed by a day hospital. Their metabolic status was carefully monitored during the month following hospitalization, with three daily determinations of blood glucose, twice a week. Insulin therapy did not need to be changed in any of the patients; the metabolic status remained constant in all diabetics. The tests were performed during this period. None of the patients had clinical features or laboratory evidence of ketosis or any signs of associated endocrine. renal, hepatic, or other intercurrent disease. Subjects with no signs of retinopathy were chosen to participate in this study. Experiments were started after adjustment of insulin therapy (dose. 24 to 50 U/d; mean, 36 U/d of the intermediate duration monocomponent insulin Metabolism, Vol 39, No 7 (July), 1990: on 668-675
CHOLINERGIC CONTROL OF GH IN DIABETICS
plus short-acting
monocomponent
insulin,
given together
669
once or
twice daily), which remained unchanged during the period of this investigation. The last injection of insulin before each test was given on the day preceding each experimental day. As indices of control of the metabolic status of these patients, blood levels of glucose were measured at 7:00 AM, I 1:OO AM, and 5:OO PM on the days preceding the tests; for each patient. a mean value for blood glucose was obtained by averaging these three determinations. On the experimental days blood samples also were taken for measurement of glycosylated hemoglobin (HbA,,). Furthermore. 24-hour urinary glucose excretion was collected at the time of the tests. Twenty-nine normal men (mean weight * SE, 71.4 + 7.2 kg; height. 1.71 i 0.08 m; BMI, 24.5; mean age, 30.3 t 1.3 years, and range, 24 to 41) without any signs of endocrine disease or family history of diabetes mellitus were studied as controls. Normal and diabetic men were tested with exercise, arginine, or GHRH in the presence or absence of pirenzepine. Preliminary
Tests
Eight normal men and eight diabetics were tested with 15.0 and 17.5 mg pirenzepine during the arginine test (four normal men and four diabetics) and the exercise test (the remaining eight subjects), in order to determine the minimal effective dose of the drug capable of modifying the GH response to arginine or exercise stimulation. In the control tests, normal saline (NaCI 0.9%) was administered instead of pirenzepine. Experiments were performed as described below. Since neither 15.0 nor 17.5 mg pirenzepine modified the GH response during arginine and exercise tests (data not presented), we decided to use higher doses of pirenzepine in the following tests. Other subjects participated in these tests in order to avoid an excessive number of experiments in the same subjects. Exercise
Test
Seven normal controls and seven diabetics were tested with exercise. Exercise tests were performed on an bicycle ergometer at an intensity of 75 W from 0 to 30 minutes. Experiments started at 8:00 AM after overnight fast. Exercise started 15 minutes after the insertion of an intravenous (IV) catheter into an antecubital vein, which was used for blood sampling. Basal blood samples were taken at time 0 (just before exercise). Further specimens were withdrawn at time 10, 20, 30. 40, 50, and 60 minutes. When the effect of pirenzepine on the GH response to exercise was tested, an equal procedure was followed, except for the injection of 20, 25, or 30 mg pirenzepine at time - 10 minutes. In the control test, an equal amount of normal saline was injected instead of pirenzepine. Control and experimental tests were performed in random order, with an interval of at least 5 days. .4rginr’ne
Test
Seven normal controls and seven diabetics were tested with arginine. At 8:00 AM of the experimental day, two indwelling catheters were inserted into forearm veins of opposite arms of subjects fasting from the previous evening and lying in the recumbent position. One catheter was used for blood sampling, the other for arginine infusion. In one test (control test), a blood specimen (0 minutes) was withdrawn before the beginning of arginine (arginine monoh,ydrochIoride, 30 g administered IV over a 30-minute period) infusion and further samples were taken at time 15, 30, 45, 60, and 90 minutes. In the other tests (experimental tests), a similar protocol was followed, except that 20,25, or 30 mg pirenzepine (Gastrozepin, Boehringer Ingelheim, Italy) was administered IV 10 minutes before arginine infusion. In the control test, an equal amount of normal saline was injected instead of pirenzepine. Control and experimental tests were performed in random order. with an interval of at least 5
days. In all subjects.
blood samples
were used for measurements
of
glucose, GH, and glucagon concentrations. Somatomedin-C (SMC), free fatty acids (FFA), and HbA,, concentrations were evaluated in the specimen taken at time 0 of all tests. GHRH
Test
Seven normal controls and seven diabetics were tested with GHRH. Experiments were performed as described for the arginine test, except that 1 fig/kg body weight GHRH (l-44, Sanofi, Toulouse, France) was given at time 0 in an IV bolus instead of arginine. All blood specimens were used for measurements of glucose and GH concentrations. SM-C. FFA, and HbAlC levels were measured in the sample taken at time 0 of all tests. During exercise, subjects breathed through a low-resistence, one-way valve connected to a P.K. Morgan measurement system (Avinton, Seattle, WA), which had been appropriately calibrated. The following parameters were measured: ventilation, frequency of breathing, tidal volume, oxygen consumption (VO?), carbon dioxide production (VCO?), and respiratory exchange ratio (R). Determination of heart rate and blood pressure were carried out by an experienced cardiologist. Heart rate was measured by auscultation over the precordium; blood pressure was evaluated with a sphygmomanometer. Assays All samples from a single subject were determined in duplicate in the same assay. GH concentrations in the serum were measured in all samples by a specific radioimmunoassay (RIA),lh using a double-antibody technique. Intraassay and interassay coefficients of variation were 3.6% and 8%. respectively. The lower limit of sensitivity was 0.5 ng/mL. Glucagon was measured by specific RIA,” using a double-antibody technique. The intraassay and interassay coefficients of variation were 5% and 6.3%. respectively. The detection limit was 14.5 pg/mL. SM-C levels were measured by RIA (double-antibody method) using kits obtained by Nichols Institute Diagnostics (San Juan Capistrano. CA), as described previously.‘x The intraassay coefficient of variation was 5% and the interassay coefficient of variation was 10%. The sensitivity of the assay was 0.1 mU/mL. FFA concentrations were determined using an enzyme-based method.19 HbA,, was assayed by high-pressure liquid chromatography after hemolysis of the red blood cells,‘O~” using reagents obtained from BIO-Rad Laboratories (Richmond, CA) (normal range. 4.15% to 9.08%). Blood glucose was measured with an IL 918 autoanalyzer (Instrumentation Laboratory, Milan, Italy), using a glucose oxidase-peroxidase procedure. Statistical analyses were performed using the Kruskall-Wallis test, analysis of variance randomized factorial block (RBF [two-way ANOVA]), ANOVA split-splot factorial design, and the Sperman’s r linear coefficient correlation, as appropriate. The areas under the curve of the glucagon and GH responses to arginine were calculated with trapezoid integration. Data are reported as mean -r SE. RESULTS
The
indices
diabetic Arginine Blood test
of control
patients
the
metabolic
in Table
status
in the
1.
Test glucose
were
levels
184.4
(time
+ I5
218.4
k 10.4 (time
minutes), values
of
are reported
minutes), and
were
+
219.4
observed
of diabetic 9.8
208.7 +45 2
patients
mg/dL k
(time 10.0
minutes), 12.1
during
(time arginine
during 0).
(time
223.8 +90
arginine
198.3 +30
+
8.8
minutes),
+ 11.6 (time minutes).
plus pirenzepine
+60
Similar tests.
COIRO ET AL
670
Table 1. Clinical and Biochemical Characteristics
Age Iv) Arginine test group (n = 7) Mean + SE 33.7 Range
i- 2.0
Range
Range
35.6
12-25
+ 2.7
f 1.7
k 3.0
35.0
lo-23
188.3
-r 2.5
38.7
5-19
HbA,,
+ 9.9’
9.0 ? 0.8’
168.4-193.5
182.0
24-50
15.1 t 1.6
24-37
AverageBlood GlucoseIfig/DLl
23-40
16.3 + 1.8
25-44
Exercise test group (n = 7) Mean + SE 28.8
InsulinDose W/24 h)
16.7 + 2.7
26-42
GHRH test group (n = 71 Mean t SE 33.2
of the Three Groups of Diabetic Patients
Diabetes Duration(yr)
8.0-9.4
k 6.3*
8.9 i 0.4’
158.4-197.4
+ 2.3
186.2
29-48
7.9-9.6
f 10.2’
9.2 i 0.7’
160.6-195.0
8.1-9.5
‘Mean of 4 test days at time 0.
30 mg pirenzepine in both groups. When 25 mg pirenzepine was administered, the inhibition observed in the control subjects (85% decrease of the mean peak response) was still significantly higher (P < .Ol) than in the diabetic patients (60%) (Fig 2). The incretory response was significantly lower in the controls than in the diabetic patients (F = 16.710. P < .OOl) (Fig 1). In both groups, the GH response to arginine was almost completely blocked with the administration of 30 mg pirenzepine (Figs 1 and 2). The basal levels of glucagon were similar in normal and diabetic subjects (Table
The basal concentration (mean of 4 test days) of SM-C (diabetics, 0.7 + 0.06 mU/mL; normals, 0.8 + 0.05) and FFA (diabetics, 0.81 k 0.09 mmol/L; normals, 0.72 f 0.08) were similar in the two groups. The results of the arginine test performed without pirenzepine and after treatment with different amount of the cholinergic antagonist are shown in Figs 1 and 2. The basal concentrations of GH were similar in the normal controls (1.5 + 0.1 ng/mL) and diabetic patients (1.8 k 0.2 ng/mL). Serum GH levels were significantly increased by arginine infusion in both groups. The mean peak concentration of GH was reached at 45 minutes in both control and diabetic subjects (P < .OOl v time 0); however, the incretory response was significantly higher in the diabetic patients than in the control group (F = 10.316, P < .002). Correlation analyses of the peak GH response to arginine in diabetics with basal and 45-minute blood glucose levels or basal HbAlC, FFA, and SM-C did not provide significant results. Administration of 20 mg pirenzepine strikingly decreased the mean peak GH response by 70% in the normal subjects, but only by 30% in the diabetic patients (P < .Ol) (Fig 2). The incretory response was significantly lower in the control group than in the diabetic patients (F = 37.906, P < .OOl) (Fig 1). Higher inhibitory effects were obtained with 25 and
2). The incretory response of glucagon was significantly higher in the diabetic patients than in the control group (F = 9.786, P -C .002) (Table 2). When GH and glucagon data of diabetic patients were considered together, correlation analyses of hormonal peaks or areas in response to arginine did not provide significant results. Administration of 20, 25 or 30 mg pirenzepine did not change the glucagon response to arginine in both normal and diabetic subjects (Table 2). Exercise Test Blood glucose levels in diabetic patients during exercise were 180.1 * 10.8 mg/DL (time 0), 188.4 + 9.7 (time + 10
Normals
0
IS
Diabetics
30
45
60
90
0
1s
JO
45
60
90
Minutes Fig 1. Serum GH response to arginine (A) alone or together with different doses of pirenrepine (P) in normal and diabetic subjects. Each point represents the mean t SE of seven observations. 0. A; +. A + P. 20 mg: n . A + P, 25 mg: 0, A + P, 30 mg.
CHOLINERGIC
CONTROL
OF GH IN DIABETICS
671
0
patients (30%) (P < .Ol) (Fig 4). The GH response to exercise was significantly lower in normal than in diabetic subjects (F = 25.265, P < .OOl) (Fig 3). Higher inhibitory effects were obtained with 25 and 30 mg pirenzepine in both groups. When exercise was performed after 25 mg pirenzepine, the GH response to exercise was significantly lower in the control than in the diabetic group (F = 9.985, P < ,002) (Fig 3). This amount of pirenzepine induced comparable percent inhibitions in diabetic and control subjects (Fig 4). In the exercise plus 30-mg pirenzepine test, GH responses to exercise (Fig 3) and percent inhibitions induced by pirenzepine were similar in diabetics and controls (Fig 4). Heart rate (diabetics: basal 75 e 8 bpm, peak 126 + 9; normals: basal 72 k 8, peak 123 t 8), mean blood pressure (diabetics: basal 97 * 5 mm Hg, peak 113 2 8; normals:basal 95 t 4, peak 109 + 7), respiratory rate diabetics: basal 12.6 k I .4 min ‘, peak 21.5 i 2.6; normals:basal 12.4 + 1.0. peak 20.3 t 2.0), ventilation (diabetics: basal 10.6 + 0.7 L/min, peak61.8 k 2.9; normals: basal 10.4 t 0.9, peak60.0 z 3.0), tidal volume (diabetics: basal 0.6 + 0.2 L. peak 2.0 k 0.4; normals: basal 0.8 IT 0.2, peak 1.8 +- 0.4). VO, (diabetics: basal 333 f 13. mL/min, peak 2,239 * 147; normals: basal 335 k 12, peak 2,234 f 150). VCO, (diabetics: basal 285 + 17 mL/min, peak 2,098 f 158; normals: basal 286 + 17, peak 2,070 + 151), R (diabetics: basal 0.86, peak 0.92; normals: basal 0.89, peak 0.93) were not significantly different between diabetic and normal subjects. Similar values were observed in both groups after treatment with 20, 25, and 30 mg pirenzepine.
-20
3 Q
-10
A.
-60 #
-80
yco.0 I
I
n.s. I
A
A+P20mg
A+P25mg
A+P30mg
Fig 2. Percentage decrease of the peak GH response (% LI GH. after subtraction of basal values) to arginine (A) induced by different amounts of pirenzepine (PI in normal (WI and diabetic (0) subjects. Each bar represents the mean + SE of seven observations.
minutes), 197.8 i 10.9 (time +20 minutes), 204.5 + 13.0 (time -t30 minutes), 215.0 k 12.4 (time +40 minutes), 221.1 :t 10.4(time +50minutes),and226.2 + 9.9 (time +60 minutes). Similar values were observed during exercise plus pirenzepine tests. The basal levels (mean of 4 test days) of SM-C (diabetics, 0.8 k 0.07 mU/mL; normals. 0.9 + 0.06) and FFA (diabetics, 0.85 * 0.10 mmol/L. normals, 0.68 2 0.07) were similar in the two groups. Basal GH levels were similar in diabetic (1.7 + 0.3 ng/mL) and normal (1.6 + 0.1 ng/mL) subjects. In both groups, exercise induced a significant increase in serum GH concentrations, with a peak response at 30 minutes (P < .OOl v time 0). The increment was significantly higher in the diabetic than in the normal subjects (F = 9.700, P < ,002) (Fig 3). The GH peak responses in diabetics were not statistically correlated with basal and 30-minute glucose concentrations and basal HbA,,, FFA, and SM-C levels. The administration of 20 mg pirenzepine significantly reduced the exercise-induced GH increase (Fig 3). However, a more marked inhibition was observed in the normal controls (60%) than in the diabetic Table 2. Glucagon Response to Arginine Administered
GHRH
Test
Blood glucose levels in diabetic patients during GHRH testing were 177.7 t 10.0 mg/dL (time 0). 178.0 i 9.8 (time + 15 minutes), 197.3 * 8.9 (time + 30 minutes), 206.0 f 7.8 (time +45 minutes), 215.9 + 8.4 (time +60 minutes), 222.9 + 10.6 (time +90 minutes), and 235.5 + 11.8 (time + 120 minutes). Similar values were observed during GHRH plus pirenzepine tests. The basal levels (mean of 4 test days) of SM-C (diabetics, 0.8 * 0.08 mU,‘mL; normals, 0.9 f 0.05) and FFA (diabetics, 0.84 +_0.09 mmol/L; normals, 0.70 k 0.09) were similar in the two groups. Basal GH levels were similar in normal (1.6 * 0.4 ng/mL) and diabetic
Alone or After Treatment
With Increasing Amounts of Pirenrepine
Glucagon(pg/mL) Treatment
Subjects
0 min
+15min
+30
+45
min
min
+60
mu”
f90
rn,”
Normals 94.7
+ 6.9
194.3
t
10.4
227.7
+ 19.4
184.7
+ 10.0
95.1
z 6.7
93.7
? 6.5
95.0
* 7.1
197.6
+ 11.8
225.4
+ 20.3
182.9
+ 11.4
93.7
i 6.0
92.8
+ 6.1
AfP25mg
93.0
‘- 5.9
200.1
f
12.0
229.5
c 19.8
180.4
+ 11.8
94.8
t 6.6
92.0
* 7.0
A+P
96.8
t 8.0
193.1
f
11.9
224.3
+ 19.7
178.8
i
12.0
95.8
t 6.8
91.9
2 8.1
A
AfP
20 mg 30 mg
Diabetics 104.0
? 6.7
245.4
+ 11.3’
293.7
? 18.0*
233.4
+ 11.8’
108.7
i- 8.9*
102.9
+ 8.2
A+P
20 mg
106.6
* 7.4
250.7
t
12.4’
296.4
+ 19.8*
240.6
? 12.0*
111.4
+ 9.7.
101.4
+ 8.7
A+P
25 mg
108.2
? 8.0
242.7
t
12.7t
289.7
+ 20.3*
230.9
t
13.0’
107.6
i 9.9t
102.5
f 7.9
A+P
30 mg
103.7
* 7.5
244.9
+ 12.0’
288.6
i 21.0*
233.6
+ 12.8”
106.5
t 9.5t
100.4
+ 8.4
A
NOTE.
Each point represents
Abbreviations: lP < .02,
A, arginine;
the mean
* SE of the observations.
P, pirenzepine.
tP < .05 between
normals
and diabetics
(Kruskall-Wallis
test).
COIRO ET AL
672
Normals
Diabetics
1
0
IO
20
30
40
50
60
0
IO
20
30
40
50
60
Mlrlutes Fig 3. Serum GH response to exercise (El alone or together with different doses of pirenzepine (P) in normal and diabetic subjects. Each point represents the mean + SE of seven observations. 0, E; +, E + P, 20 mg: n , E + P, 25 mg, 0, E + P, 30 mg.
(1.9 + 0.4) subjects. In both groups, GHRH induced a significant increase in serum GH concentrations, with a peak response at 15 minutes (P < ,001 v time 0). GH responses in the two groups were similar (Fig 5). The administration of 20 mg pirenzepine significantly reduced the GHRH-induced GH increase (Fig 5). However, normal controls showed a more marked inhibition (50%) than diabetic patients (22%) (P < .02) (Fig 6). The GH response to GHRH was significantly lower in normal controls than in diabetics (F = 29.899, P < ,001) (Fig 5). When 25 mg pirenzepine was administered, the inhibition obtained in the control subjects (70% decrease of the mean peak response) was significantly higher than in the diabetic patients (45%) (P < .02) (Fig 6). Thirty milligrams pirenzepine produced a similar and almost complete blockade of the GH response to GHRH in both groups (Figs 5 and 6). At all doses, pirenzepine induced a similar transient impairment of visual accommodation in all subjects. 0
-20 %
-40
bQ
-60
4
-80 11.5. -100
!
E
Et P20mg
E+P25mg
E * P3Omg
Fig 4. Percent decrease of the peak GH response 1% A GH. after subtraction of basal values) to exercise (E) induced by different amounts of pirenzepine (P) in normal (W) and diabetic (ml subjects. Each bar represents the mean + SE of seven observations.
DISCUSSION
The results of the present study confirm that in patients with type I diabetes mellitus there is a higher GH responsiveness to arginine infusion and physical exercise than in normal subjects. Abnormal GH responses in diabetes mellitus have been attributed to poor metabolic control.‘.“.” Our data do not support this hypothesis, since no correlations were found between peak GH responses to exercise or arginine and the metabolic status of our patients (blood glucose, FFA, and HbA,, levels). In addition, SM-C levels were not correlated with peak GH responses. In agreement with previous reports,*‘-” diabetic patients presented a higher glucagon increase than normal controls during arginine infusion. Glucagon is a well-known stimulator of GH release.‘7”9 and thus, the higher glucagon increase in diabetics might have induced the increased GH response to arginine. This possibility is unlikely, because no correlation was observed between glucagon and GH responses to arginine. However, if glucagon were responsible for the higher GH response to arginine in diabetics, it should have exerted this effect through muscarinic cholinergic mediation. In fact, in the presence of pirenzepine, the GH response to arginine was blunted, even though the glucagon increase remained unmodified. In both control and diabetic subjects, pirenzepine administered at concentrations ranging from 20 to 30 mg produced a dose-related decrease of the GH responses to stimulation with exercise or arginine. The inhibitory effect of pirenzepine was significantly smaller in diabetic than in normal subjects when lower doses of the drug were administered (20 and 25 mg in the arginine test and 20 mg in the exercise test), but it was similar in both groups when a saturating amount of the cholinergic antagonist (30 mg) was administered. The higher dose of pirenzepine necessary for suppression in diabetics might be interpreted as an effect of the altered hormonal milieu conditioning hyperresponsiveness to arginine and exercise in these subjects; for example, it might be attributed to the simultaneous higher glucagon response during argin-
CHOLINERGIC
CONTROL
OF GH IN DIABETICS
673
Normals l-
Diabetics
pa
,-
I I-
\i
\ I
0
15
30
I
45
I
1
60
90
1
0
120
15
30
45
60
90
120
Minutes Fig 5.. Serum GH response to GHRH alone or together with different doses of pirenzepine (PI in normal and diabetic subjects. Each point represents the mean + SE of seven observations. 0, GHRH; +, GHRH + P, 20 mg; n , GHRH + P, 25 mg, 0, GHRH + P, 30 mg.
ine infusion. This hypothesis was not confirmed by our additional tests with GHRH. In fact, even though GHRH induced similar GH responses in normal and diabetic subjects, the differences in pirenzepine action between groups were similar to those observed in the arginine and exercise tests. Taken together, these results suggest the existence of intrinsic changes in the cholinergic system of type I diabetics. The shift of the dose-response curve in the diabetic subjects indicates a lower sensitivity of the cholinergic system to muscarinic receptor blockade. However, we were unable to detect a change in diabetics of the minimal concentration of pirenzepine capable of inducing a statistically significant inhibition (20 mg in both control and diabetic subjects). The possible existence of different mini0
-40 3 -60
cl Be
-80
n.s
-100
GHRH
GHRH+ P 20mg
GHRH+ P 25mg
GHRH+ P 30mg
Fig 6. Percent decrease of the peak GH response (% A GH, after subtraction of basal values) to GHRH induced by different amounts of pirenzepine (PI in normal (ml and diabetic (al subjects. Each bar represents the mean * SE of seven observations.
ma1 effective doses in the range between IS and 20 mg pirenzepine might be masked by the poor sensitivity of our in vivo model. In light of our present results, the higher GH responsiveness of the diabetic patients to arginine and exercise might be attributed to an increased activity of cholinergic neurons. This phenomenon might be an aspect of a more general disorder affecting the neuroendocrine control of GH secretion in type 1 diabetic subjects. Particularly, the GH responses to exercise and arginine are also regulated by adrenergic stimuli.7~‘0 and thus it is likely that both cholinergic and adrenergic systems work in concert to tonically influence exercise- and arginine-induced GH secretion. In view of the well-known adrenergic alterations affecting type I diabetics3’-j5 our results might reflect a modification in diabetics of the possible interplay between adrenergic and cholinergic stimuli. The reduced effect of pirenzepine in our diabetic patients might be explained by changes in cholinergic muscarinic receptor number or sensitivity. Cholinergic receptors are present both at hypothalamic and pituitary level.3h~3” As pirenzepine is unable to cross the blood brain barrier (BBB)39 and our patients were not affected by diabetic retinopathy and thus presumably had a normal BBB permeability, cholinergic alterations in diabetics might be supposed at the pituitary level or in hypothalamic structures outside the BBB (ie, the median eminence). At present, there is no evidence for a direct cholinergic modulation of GH secretion at the pituitary level. In fact, atropine administered in vivo has been found capable of blocking the GHRH-induced GH secretion in humans, whereas atropine added in vitro was unable to change the GH response to GHRH in bovine pituitary cell culture.4” Changes at the level of the median eminence might be supposed. It has been reported that acetylcholine inhibits the release of somatostatin from the hypothalamus in vitro
674
CON0
through a muscarinic mechanism,” and thus, pirenzepine might enhance endogenous somatostatin release, blocking the GH response to arginine, exercise, and GHRH. In light of this hypothesis, our results might indicate that in diabetes mellitus there is a cholinergic receptor alteration affecting the mechanism controlling hypothalamic somatostatin re-
lease. Further studies are needed to substantiate esis.
ET AL
this hypoth-
ACKNOWLEDGMENT
We thank Dr Claudio Nava (Sanofi Midy, Milan Italy) for generously supplying GHRH.
REFERENCES I, Johansen K. Hansen AP: High 24-hour level of serum growth hormone in juvenile diabetics. Br Med J 2:356-357, 1969
17. Henquin JC. Malvaux P, Lambert AE: Glucagon immunoassay using polyethylene glycol to precipitate antibody-bound hormone. Diabetologia 10:61-68, 1974
2. Molnar GD. Taylor WF, Langworthy A, et al: Diurnal growth hormone and glucose abnormalities in unstable diabetics: Studies of ambulatory fed subjects during continuous blood glucose analysis. J Clin Endocrinol Metab 34:837-846, 1972
18. Furlanetto RW, Underwood LE. Van Wyk JJ, et al: Estimation of somatomedin C levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest 60:648-657, 1977
3. Press M, Tamborlane WV, Sherwin RS: Importance of raised growth hormone levels in mediating the metabolic derangements of diabetes. N Engl J Med 31:810-815. 1984
19. Mizuni K. Toyosato M, Yabumoto method for calorimetric determination Biochem 108:6-IO, 1980
4. Vigneri R, Squatrito S, Pezzino V, et al: Growth hormone levels in diabetes: Correlation with the clinical control of the disease. Diabetes 25: 167- 172, 1976
20. Koenig RJ. Cerami A: Synthesis of hemoglobin A,, in normal and diabetic mice: Potential model of basement membrane thickening. Proc Nat1 Acad Sci 72:3687-3691, I975
5. Yde H: The immunoreactive growth-hormone in serum from patients with various types of diabetes mellitus. Acta Endocrinol 64~339-346. 1970
21. Koenig RJ, Araujo DC, Cerami A: Increased in diabetic mice. Diabetes 25: l-5, 1976
6. Hansen AP: Abnormal serum growth hormone response exercise in juvenile diabetics. J Clin Invest 49:1467-1478, 1970
to
S, et al: A new enzymatic of free fatty acid. Anal
hemoglobin
A,,
22. Hansen AP, Johansen K: Diurnal patterns of blood glucose. serum free fatty acids, insulin, glucagon and growth hormone in normals and juvenile diabetics. Diabetologia 6:27-33, 1970
7. Hansen AP: The effect of adrenergic receptor blockade on the exercise-induced serum growth hormone rise in normals and juvenile diabetics. J Clin Endocrinol Metab 33:807-812, 1971
23. Johansen K, Hansen AP: Diurnal serum growth levels in poorly and well-controlled juvenile diabetics. 20~239-245, 1971
8. Burday SZ, Fine PH. Schalch DS: Growth hormone secretion in response to arginine infusion in normal and diabetic subjects: Relationship to blood glucose levels. J Lab Clin Med 71:897-911, 1968
24. Aguilar-Parada E. Eisentraut AM, Unger RH: Pancreatic glucagon secretion in normal and diabetic subjects. Am J Med Sci 257:415-419, 1969
9. Arends J, Wagner ML, Willms BL: Cholinergic muscarinic receptor blockade suppress arginine- and exercise-induced growth hormone secretion in type I diabetic subjects. J Clin Endocrinol Metab 66:389-395, 1988 IO. Chiodera P. Coiro V. Speroni G. et al: The growth hormone response to thyrotropin-releasing hormone in insulin-dependent diabetics involves a cholinergic mechanism. J Clin Endocrinol Metab 59:794-797, 1984
I I. Delitala G, Tomasi P. Virdis R: Neuroendocrine human growth hormone secretion. Diagnostic tion. J Endocrinol Invest 11:441-464, 1988
regulation of and clinical applica-
12. Pietschmann P, Schernthaner G, Luger A: Effect of cholinergic muscarinic receptor blockade on human growth hormone (GH)-releasing hormone (I-44)-induced GH secretion in acromegaly and type I diabetes mellitus. J Clin Endocrinol Metab 63:389393. 1986 13. Giampietro 0, Ferdeghini M. Miccoli R, et al: Effect of growth hormone-releasing hormone and clonidine on growth hormone release in type 1 diabetic patients. Horm Met Res 19:636-641, 1987 14. Kopelman PG, Mason AC, Noonan K. et al: Growth hormone response to growth hormone releasing factor in diabetic men. Clin Endocrinol28:33-38, 1988 15. Press M, Tamborlane response to growth hormone 33:804-806, 1984
WV, Thorner MO, et al: Pituitary releasing factor in diabetes. Diabetes
16. Schalch DS, Parker ML: A sensitive double antibody immunoassay for human growth hormone in plasma. Nature 203:1141I142,1964
25. Aronoff SL. Bennet stimulated hyperglucagonemia 407, 1976
hormone Diabetes
PH, Rushforth NB, et al: Argininein diabetic subjects. Diabetes 25:404-
26. Unger RH, Aguilar-Parada E, Muller WA. et al: Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837-848, 1970 27. Mitchell ML, Byrne MJ. Silver J: Growth hormone release by glucagon. Lancet 1:289-290, 1969 28. Rao RH. Spathis GS: lntramuscolar glucagon as a provocative stimulus for the assessment of pituitary function: Growth hormone and cortisol responses. Metabolism 37:659-663. 1987 29. Wieland RG, Hallberg MC, Zorn EM: Growth hormone response to intramuscolar glucagon. J Clin Endocrinol Metab 37:329-330, 1973 30. Buckler JMH, Bold AM, Taberner M, et al: Modification of hormonal responses to arginine by cu-adrenergic blockade. Br Med J 3:153-154, 1969 31. Bolli G, Compagnucci P, Cartechini MG, et al: Urinary excretion and plasma levels of norepinephrine and epinephrine during diabetic ketoacidosis. Acta Diabetol Lat 16:157-167, 1979 32. Christensen NJ: Plasma norepinephrine and epinephrine in untreated diabetics, during fasting and after insulin administration. Diabetes 23: l-8. 1974 33. Robertson RP. Halter JB, Porte DJ Jr: A role for alphaadrenergic receptors in abnormal insulin secretion in diabetes mellitus. J Clin Invest 57:791-795, 1976 34. Speroni G, Ceda GP, Capretti L, et al: Clonidine and GH secretion in insulin dependent diabetes (IDD). Horm Metab Res 15146-47. 1983 35. Tamborlane
WV, Sherwin
RS, Koivisto V, et al: Normaliza-
CHOLINERGIC CONTROL OF GH IN DIABETICS
tion of the growth hormone and catecholamine response to in juvenile-onset diabetic subjects treated with a portable infusion pump. Diabetes 28:785-788. 1979 36. Burt DR. Taylor RL: Muscarinic receptor binding anterior pituitary. Neuroendocrinology 30:344-349. 1980 37. Schaeffer JM, Hsueh AJW: Acetylcholine in the rat pituitary gland. Endocrinology 106:1377-1381. 1980 38. Taylor RL, Burt DR: Pituitary cell cultures contain inic receptors. Em J Pharmacol 65:305-308, 1980
675
exercise insulin in sheep anterior muscar-
39. Hammer R, Koss FW: The pharmacokinetic profile of pirenzepine. Scan J Gastroenterol 14:l-20. 1979 (suppl57) 40. Casanueva FF. Villanueva L, Dieguez C, et al: Atropine blockade of growth hormone (GH)-releasing hormone-induced GH secretion in man is not exerted at pituitary level. .J Clin Endocrinol Metab62:186-191, 1986 41. Richardson SB, Hollander CS. D’Eletto R, et al: Acetylcholine inhibits the release of somatostatin from rat hypothalamus in vitro. Endocrinology 107: 122- 129. 1980