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Resumo
Em pacientes com diabetes mellitus tipo 1 (DM1) foram observadas respostas
normais ou aumentadas de GH após estímulos. O eixo hipotálamo" hipófise"
adrenal tem sido menos estudado. A ghrelina é um secretagogo de GH que
também aumenta os níveis de ACTH e de cortisol, de forma semelhante ao
GHRP"6. Não existem estudos sobre os efeitos da ghrelina em pacientes com
DM1. O objetivo do presente trabalho foi avaliar as respostas do GH, ACTH e
cortisol à ghrelina e ao GHRP"6 em 9 pacientes com DM1 (HbA1c: 11.7 ± 1,3%;
média ± SE) e em 9 indivíduos normais. A liberação de GH induzida por GHRH
também foi estudada. A média dos níveis basais de GH (;g/L) foi maior nos
pacientes com DM1 (3.5 ± 1.2) do que nos controles (0.6 ± 0.3). Analisando AASC
GH (;g/L.120min), não foram observadas diferenças significantes entre os
diabéticos (ghrelina: 3.148 ± 427; GHRP"6: 1428 ± 299; GHRH: 885 ± 184) e os
controles (ghrelina: 3228 ± 1036 ; GHRP"6: 1271 ± 217; GHRH: 643 ± 178). Em
ambos os grupos, a liberação de GH induzida pela ghrelina foi maior do que após
GHRP"6 e GHRH. Houve uma tendência (p = 0.055) à elevação dos níveis de
cortisol basal (;g/dL) nos pacientes com DM1 (11.7 ± 1.5) comparado aos
controles (8.2 ± 0.8). Não foram observadas diferenças significantes nos valores
de AASC cortisol (;g/dL.90min) entre os grupos após ghrelina (DM1: 303 ± 106;
controles: 467 ± 86) e GHRP"6 (DM1: 135 ± 112; controles: 187 ± 73). A média de
valores basais de ACTH (pg/mL) foi semelhante nos diabéticos (19.9 ± 3.4) e nos
controles (14.5 ± 2.3). Não foram observadas diferenças nos valores de AASC
ACTH (pg/mL.90min) entre os grupos após ghrelina (DM1: 1372 ± 771; controles:
1394 ± 327) e GHRP"6 (DM1: 257 ± 291; controles: 423 ± 211). Em resumo, os
pacientes com DM1 têm resposta normal do GH à ghrelina, GHRP"6 e GHRH. A
liberação de ACTH e cortisol após ghrelina e GHRP"6 também é semelhante aos
controles. Nossos resultados sugerem que a hiperglicemia crônica do DM1 não
Effects of ghrelin, GH releasing peptide 6 (GHRP 6) and GHRH on GH, ACTH
and cortisol release in type 1 diabetes mellitus
! " # $ % & ! ' %
(
Division of Endocrinology, Universidade Federal de São Paulo, UNIFESP"EPM,
São Paulo, Brazil
Abbreviated title:Ghrelin and type 1 diabetes mellitus
Keywords:ghrelin, GH, ACTH, cortisol, type 1 diabetes mellitus
Corresponding author:
Larissa Bianca Paiva Cunha de Sá
Rua Pedro de Toledo, 910
04039"002 – São Paulo"SP – Brazil
Phone/fax: 55"11"55748432
Abstract
In type 1 diabetes mellitus (T1DM), GH responses to provocative stimuli are
normal or exaggerated while the hypothalamic"pituitary"adrenal axis has been less
studied. Ghrelin is a GH"secretagogue which also increases ACTH and cortisol
levels, similarly to GHRP"6. Ghrelin´s effects in patients with T1DM have not been
evaluated. We, therefore, studied GH, ACTH and cortisol responses to ghrelin and
GHRP"6 in 9 patients with T1D1 (HbA1c: 11.7±1.3%; mean±SE) and 9 control
subjects. GHRH" induced GH release was also evaluated. Mean basal GH levels
(Rg/L) were higher in T1DM (3.5±1.2) than in controls (0.6±0.3). Analyzing ∆AUC
GH values (Rg/L.120min), no significant differences were observed in T1DM
(ghrelin: 3148±427; GHRP"6: 1428 ±299; GHRH: 885 ±184) compared to controls
(ghrelin: 3228±1036; GHRP"6: 1271±217; GHRH: 643±178). In both groups,
ghrelin"induced GH release was higher than after GHRP"6 and GHRH. There was
a trend (p=0.055) to higher mean basal cortisol values (Rg/dL) in T1DM (11.7±1.5)
compared to controls (8.2±0.8). No significant differences were seen in ∆AUC
cortisol values (Rg/dL.90min) in both groups after ghrelin (DM1:303±106; controls:
467±86) and GHRP"6 (DM1:135±112; controls: 187±73). Mean basal ACTH values
(pg/mL) were similar in T1DM (19.9±3.4) and controls (14.5±2.3). No differences
were seen in ∆AUC ACTH levels (pg/mL.90min) in both groups after ghrelin
(DM1:1372±771; controls: 1394±327) and GHRP"6 (DM1: 257±291; controls: 423
±211). In summary, patients with T1DM have normal GH responsiveness to
ghrelin, GHRP"6 and GHRH. ACTH and cortisol release after ghrelin and GHRP"6
is also similar to controls. Our results suggest that chronic hyperglycemia of T1DM
does not interfere with GH, ACTH and cortisol releasing mechanisms stimulated by
Introduction
Growth hormone (GH) secretion is modulated by an interplay between
growth hormone"releasing hormone (GHRH) and somatostatin (SRIF). GH
secretagogues (GHS) might also have a role in this process, acting at both pituitary
and hypothalamic receptors ) * + ,-./0 " * + ,--10 % + ,--23.
Ghrelin, the endogenous ligand of GHS"receptor (GHS"R), was discovered in the
stomach, but is also present in the hypothalamus, mainly in the arcuate nucleus
)4 5 6 + ,---0 7 + 89980 ( + 899/3. The chemical
structure of this acylated peptide is different from GHS )4 5 6 + ,---3. Ghrelin
and GH"releasing peptide"6 (GHRP"6), a GHS, induce GH, ACTH and cortisol
release )% + 899,3. Their main site of action is the hypothalamus, as
demonstrated in hypothalamic"pituitary disconnection studies, in which these
effects are reduced or abolished ) 7 + ,--:0 " ; ( + ,--10 6 +
,---0 7 + 899<3. It has been shown that GHRH and ghrelin/GHS act through
different receptors and intracellular mechanisms at pituitary level. Ghrelin might
stimulate several interdependent pathways of GH release, as demonstrated
recently) = + 899<0 4 6 + 89923.
Ghrelin and GHS are also able to stimulate ACTH and cortisol release in
humans, but the magnitude of ACTH response with ghrelin is higher )> ; ( +
89990 % + 899,0 + 89913.This effect is due to an hypothalamic action
of these peptides, since they do not increase ACTH release from pituitary
fragments ) + ,--<0 4 5 6 + ,---3, and normal corticotrophs do
not express GHS"R )4 ! + 899,3. In hypothalamic" pituitary disconnection,
the effects of GHS on cortisol release are markedly reduced )" ; ( + ,--10
6 + ,---3. Although controversial, GHS and ghrelin stimulate ACTH
secretion in humans probably through hypothalamic arginine vasopressin (AVP)
releasing"pathways)4 ! + ,--- 0 + 899:3.
In patients with type 1 diabetes mellitus (T1DM) basal growth hormone
(GH) levels are either normal ) ? + ,-2-0 4 ( + ,--/0 + ,--:0
! + ,--10 6 + ,--10 $ + ,--20 + ,--.0 +
,-.,0 + ,-./0 % 7 + ,-.-0 ( + ,--/0 + ,--23, depending
on glycemic control, and insulin"like growth factor"I (IGF"I) levels are normal)"
+ ,-.,0 $ + ,--20 + 899/3 or reduced)% 7 + ,-.-0 (
+ ,--/0 ! + ,--10 6 + ,--10 + ,--.0 % @ + 899<0
" 6 + 899/0 + 899:3. It has been suggested that the enhanced GH
secretion might be due to a decrease in IGF"I feedback on the hypothalamic"
pituitary unit)" ( + ,-..3.
There are controversial findings in the literature about stimulated GH values
in patients with T1DM. Both normal and enhanced responsiveness to several
stimuli, such as GHRH and hexarelin have been reported) ? + ,-2-0
+ ,-./0 A + ,-..0 B 6 + ,--<0 + ,--<0 4 ( + ,--/0 +
,--:0 + ,--10 ! + ,--10 6 + ,--10 A + ,--.0
% 6# + ,---0 + ,---0 & + 89990 % @ + 899<0 + 899/0
+ 89910 + 89923. We have previously shown that GH
responses to GHRH and GHRP"6 are similar in T1DM and in healthy subjects
)$ + ,--23. However, Catalina et al. reported enhanced GH responses to
these peptides in diabetic patients with higher HbA1clevels) + ,--.3.
It has been suggested that patients with T1DM have hyperactivity of the
hypothalamic"pituitary"adrenal (HPA) axis. Although controversial, it has been
shown that these patients have an increase in circulating cortisol values. A change
in the pattern of 24 hours cortisol secretion has also been described and
glucocorticoid negative feedback is impaired )" + ,-./0 @@ + ,--<0
( + ,--<3. The hyperactivity of HPA axis in T1DM could be due to activation of
CRH/AVP hypothalamic neurons, changes in ACTH release from corticotrophs
and/or alteration of adrenal sensitivity ) + 8998 0 + 899.3.
Interestingly, in experimental studies hyperglycemia activates POMC gene
expression )% ! + 89923 and enhances ACTH release from normal anterior
pituitary cells) + ,--.3+
There are no data in the literature about the effects of ghrelin on GH, ACTH
Therefore, the aim of our study was to evaluate GH, ACTH and cortisol
release after the administration of ghrelin and GHRP"6 in a group of patients with
T1DM with poor metabolic control. GHRH" induced GH release was studied
Subjects and methods
!5
Nine patients (5 men and 4 women) with type 1 diabetes were studied. Their
mean age was 24.1 ± 1.5 years (mean ± SE; range: 18"30), with a mean body
mass index (BMI) of 21.2 ± 0.8 kg/m2 (range: 18.1 "24.5). The duration of diabetes
was 5.6 ± 1.3 years (range: 0"10). Mean hemoglobin A1c (HbA1c) level at the time
of evaluation was 11.7 ± 1.3% (normal range: 4.6 "6.5%). All patients had normal
thyroid and renal function and were receiving one to four injections of insulin daily.
None had clinical or laboratory evidence of nephropathy or neuropathy. Two of
them were taking angiotensin"converting enzyme inhibitors. None of the patients
had other associated diseases. Patients with hypoglycemia episodes were
excluded from the study group.
Nine normal subjects (6 men and 3 women) were also studied as a control
group. Their mean age was 28.4 ± 1.5 years (range: 20"35) and their mean BMI
was 23.3 ± 0.8 kg/m2 (range: 18.8"25.9). Mean HbA1c was of 5.0 ± 0.1%. All
subjects had normal thyroid function and were free of any medication at the time of
the study. The women were tested in the early follicular phase of the menstrual
cycle.
(
The experimental protocol was approved by the Ethics Committee of
Universidade Federal de São Paulo, and all subjects were studied after giving
informed consent. The tests were performed after an overnight fast and the
subjects remained recumbent throughout it. The patients did not receive insulin on
the morning of the study. Each subject underwent three tests, randomly, with an
interval of at least 48 h between them. Forty"five minutes before starting the test,
an indwelling catheter was inserted into an antecubital vein and kept patent by
slow 0.9% saline infusion. After the first blood sample each subject received
ghrelin (Neosystem, Strasbourg, France) at a dose of 1 Rg/kg, GHRP"6 (Bachem,
San Carlos, USA) at the same dose, or GHRH(1"29)NH2 (Clinalfa, Laufelfingen,
15 minutes until 120 minutes for hormonal determinations. Baseline blood samples
were also obtained for IGF"I and glucose measurements.
% (
Serum GH was measured in duplicate by a two"site monoclonal antibody
immunofluorometric assay )$ + ,--93. The sensitivity of the method is 0.02
Rg/L, with mean intra" and inter"assay coefficients of variation (CV) of 8.8% and
13.9% respectively. An immunochemiluminometric assay (DPC, Los Angeles,
USA) was used to measure plasma ACTH. The sensitivity of the method is 5
pg/mL, with mean intra" and inter"assay CV of 2.8% and 3.6% respectively. Serum
cortisol levels were measured in duplicate by a fluoroimmunoassay assay (Wallac,
Turku, Finland), with sensitivity of 0.2 Rg/dL, and mean intra" and inter"assay CV of
6.2% and 8.2% respectively. IGF"I levels were determined by an
immunochemiluminometric assay (DPC, Los Angeles, USA) with sensitivity of 20
ng/mL and mean intra" and inter"assay CV of 3.8% and 5.4% respectively. Plasma
glucose levels were determined by the hexokinase method (Advia 1650, Bayer,
USA). HbA1c levels were measured by high"performance liquid chromatography
(Tosoh, South San Francisco, USA).
% (
Friedman´s analysis of variance was performed to compare GH, ACTH and
cortisol levels after the injection of each peptide. Wilcoxon signed rank sum test
was used for comparisons of ACTH and cortisol values within the same group.
Mann"Whitney rank sum test was performed for comparisons between patients and
controls. The mean basal levels were calculated using all individual values
obtained before the injection of each peptide. The responses were also analyzed
by the ∆ area under the curve (∆AUC), which was calculated by trapezoidal
integration with subtraction of basal levels. Spearman´s correlation coefficient was
calculated when appropriate. For statistical purposes, undetectable GH values
were considered to be equal to 0.02 Rg/L. Results are shown as mean ± SE. P <
Results
There were no differences in age and BMI between the two groups. HbA1c
values were significantly higher in T1DM patients.
Mean basal GH levels (Rg/L) were 3.5 ± 1.2 in diabetic patients and these
values were significantly higher than those of controls (0.6 ± 0.3). IGF"I levels
(ng/mL) were similar between the two groups (T1DM: 165.7 ± 12.9; controls: 179.3
± 20.6). There was a trend to higher mean basal cortisol levels (Rg/dL) in diabetic
patients (11.7 ± 1.5) compared to controls (8.2 ± 0.8) (P=0.06). Mean basal ACTH
values (pg/mL) were 19.9 ± 3.4 in T1DM and did not differ significantly from those
observed in controls (14.5 ± 2.3). Mean basal glucose values (mg/dL) were higher
in T1DM patients (200 ± 22) than in controls (84 ± 2).
" 7 )A , 83
In controls mean peak GH (Rg/L) and ∆AUC (Rg/L.120min) values after
ghrelin administration were 57.6 ± 18.5 and 3228 ± 1036 respectively. This
response was higher than that obtained after GHRP"6 (24.4 ± 2.9; 1271 ± 217) and
GHRH (9.3 ± 2.0; 643 ± 178), which were also significantly different. In the diabetic
group GH values after ghrelin (peak: 74.6 ± 10.3; ∆AUC: 3148 ± 427) were higher
than those observed after GHRP"6 (peak: 40.3 ± 4.4; ∆AUC: 1428 ± 299) and
GHRH (peak: 21.3 ± 4.3; ∆AUC: 885 ± 184). Peak GH values after GHRP"6 were
higher than that obtained after GHRH, but ∆AUC values were similar.
When T1DM patients were compared with the control group, no differences
were found in GH responsiveness to ghrelin and GHRH. Peak GH response to
GHRP"6 was higher in T1DM patients, but no significant differences were seen in
7 )A , 83
In controls, there was a trend (p=0.055) to higher responses in cortisol
release with ghrelin (peak: 16.0± 1.3 Rg/dL and ∆AUC: 467± 86 Rg/dL.90 min)
compared to GHRP"6 (peak: 13.1 ± 0.8; ∆AUC: 187 ± 73). In T1DM patients, there
were no differences between ghrelin (peak: 18.2 ± 1.2; ∆AUC: 303 ± 106) and
GHRP"6" induced (peak: 16.7 ± 0.9; ∆AUC: 135 ± 112) cortisol release. Cortisol
responses to ghrelin were similar between diabetic patients and controls. Peak
cortisol responses to GHRP"6 were higher in T1DM but no significant differences
were observed when ∆AUC values were analyzed.
% >" 7 )A , 83
In controls mean peak ACTH (pg/mL) and ∆AUC (pg/mL.90min) values after
ghrelin (53.2 ± 10.2; 1394 ± 327) were higher than those of GHRP"6 (28.4 ± 7.6;
423 ± 211). In diabetic patients mean peak ACTH and ∆AUC values after ghrelin
(68.2 ± 19.6 and 1372 ± 771) were higher than observed after GHRP"6 (35.8 ± 6.1;
257 ± 291), but did not reach statistical significance. When T1DM patients and
controls were compared, ACTH responses to ghrelin and GHRP"6 were similar.
One or several of these symptoms (hunger, heat sensation, nausea,
transient facial flushing and sleepiness) were reported in a few patients and
Fig. 1 Mean peak GH(A), cortisol(B) and ACTH(C) values after ghrelin, GHRP
6 and GHRH administration in diabetic patients and in control subjects (mean
±SE).
DM
0 30 60 90 120
0 10 20 30 40 50 60 70 GHRELIN GHRP"6 GHRH G H ( g /L ) A Controls
0 30 60 90 120
0 30 60 90
5 10 15 20 B C o rt is o l( g /d L )
0 30 60 90
0 30 60 90
10 20 30 40 50 60 70 C Time(min) A C T H (p g /m L )
0 30 60 90
Fig. 2 Mean ∆AUC GH(A), cortisol(B) and ACTH(C) levels after ghrelin,
GHRP 6 and GHRH administration in diabetic patients and in control subjects
(mean ± SE; a, P<0.05 vs. ghrelin; b, P<0.05 vs. ghrelin and GHRP 6; c,
P=0.055 vs ghrelin).
Discussion
In our study mean basal GH levels were significantly higher in diabetic
patients compared to normal subjects, in agreement with previous reports)"
+ ,-.,0 + ,--80 ( + ,--/0 + ,--23. This might be related to
metabolic control, as in our study HbA1clevels were higher than observed in earlier
reports which failed to show an increase in basal GH values. Moreover, mean IGF"
I levels were unchanged in our patients, which suggests that reductions in
circulating levels of this peptide are not related to enhanced GH secretion. Our
findings could reflect the increased frequency of GH pulses and the elevation of
interpulse GH concentrations observed in these patients )% 7 + ,-.-3.
Moreover, it has been shown that circulating GH clearance is reduced in T1DM
) + 89983, which could contribute, at least in part, for the increased GH
values.
Interestingly, despite their poor metabolic control, GH release after ghrelin in
patients with T1DM was similar to that seen in normal subjects. Also, like
previously reported by us in patients with better glycemic control )$ +
,--23, there were no differences in GH release after GHRP"6 and GHRH in this
group of patients with high HbA1c values, which is in contrast to the results of
Catalina et al.. The lack of effect of metabolic control has been also been
previously demonstrated for GHRH"induced GH release in patients with T1DM
) + ,--83. Moreover, in some of the studies which found enhanced GH
release after GHS or GHRH, basal GH values were much higher in diabetes (>5
fold) compared to controls, despite lack of statistical significance. This could
eventually contribute to an increase in AUC values and be interpreted as enhanced
responsiveness. The normal GH responses to ghrelin, GHRP"6 and GHRH in
diabetic patients could be considered inappropriate, since it has been reported that
oral glucose administration blunts the GH response to ghrelin/GHS and GHRH in
normal subjects ) + ,-./0 + ,-.:0 + ,--:0 +
In anorexia nervosa (AN), another condition associated with high GH levels,
a reduced GH response to ghrelin was observed, despite higher GH
responsiveness to GHRH ) + 899/0 5 + 89913. Therefore, chronic
malnutrition has a diffferent effect on ghrelin"induced GH release than chronic
hyperglycemia.
Interestingly, it has been shown that insulin decreases GHRH"R and GHS"R
mRNA in pituitary tissue of baboons . Therefore, theoretically, a decrease in
endogenous insulin could enhance pituitary expression of GHRH"R and GHS"R,
) # + 89913 and eventually increase GH responsiveness to both GHRH and
GHS. However, as our results demonstrate that the pathways of GH release
stimulated by GHRH and GHS/ghrelin are not altered by persistent hyperglycemia,
this hypothesis seems unlikely in humans. Moreover, patients with T1DM were
receiving treatment with insulin.
In our diabetic patients a trend to higher basal cortisol values was observed.
This is in agreement with previous reports of mildly elevated circulating cortisol
levels and increased 24h urinary free cortisol values described in diabetic patients
) ( + ,--<0 @@ + ,--<3. Moreover, it has also been shown that the
circadian pattern of cortisol secretion is altered in these patients with increases in
trough values)C @6 + ,-2,0 6 + ,-./3.
Interestingly, it has been demonstrated both an increase and a reduction in
adrenal sensitivity in streptozotocin"induced diabetes in animals, together with
enlargement of the adrenal glands) + 8998 0 + 899.3.
However, in our study cortisol responsiveness to ghrelin and GHRP"6 was
similar in T1DM and controls, which suggests that adrenal responsiveness to these
peptides is unchanged in patients with T1DM. This is in agreement with previous
reports showing normal cortisol responsiveness to metyrapone, lysine vasopressin
and ACTH in diabetic patients) + ,---0 6 + ,--10 4 ( + ,--/3.
In our study, basal ACTH levels in patients with T1DM were similar to those
of controls, like previously reported ) @@ + ,--<0 4 ( + ,--/3. This is in
contrast with experimental studies which have shown an increase in basal ACTH
responsiveness to ghrelin and GHRP"6 in patients with T1DM and controls,
confirming earlier reports with other stimuli)A + ,-..0 @@ + ,--<0 4 (
+ ,--/0 % 6# + 899,3. It has been demonstrated that GHRP"6 stimulates
AVP release from hypothalamic fragments )4 ! + ,---!3, while
ghrelin enhances AVP, corticotrophin"releasing hormone (CRH) and neuropeptide
Y (NPY) release )C + 89983, with a predominant action on AVP secretion
) @ + 899<3. Although controversial, it has been shown in experimental models
of diabetes that hypothalamic CRH mRNA levels are increased and AVP mRNA
values are unchanged ) * @ + ,--20 + 8998!0 + 899.3.
Therefore, if also true for humans, the lack of changes in AVP mRNA in diabetes
could eventually explain our findings as GHS/ghrelin stimulate ACTH secretion in
humans probably through AVP pathways)4 ! + ,--- 0 + 899:3.
In summary, patients with T1DM have normal GH responsiveness to ghrelin,
GHRP"6 and GHRH. ACTH and cortisol release after ghrelin and GHRP"6 are also
similar to controls. Our results suggest that chronic hyperglycemia of DM1 does not
interfere with GH, ACTH and cortisol"releasing mechanisms stimulated by these
peptides. Further studies are necessary to elucidate these findings.
Acknowledgements
We would like to thank Dr. Carlos Dieguez for the initial encouragement and
help. We are grateful to Ap. Filomena P. Machado and Walkiria Miranda for the
technical assistance. We would like to thank FAPESP (Fundação de Amparo à
Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de
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Sumário e Conclusões
Os pacientes com diabetes mellitus tipo 1 tem resposta normal do GH após
a administração de ghrelina, GHRP"6 e GHRH. A liberação de ACTH e cortisol
após a injeção de ghrelina e GHRP"6 também é semelhante aos controles.
Nossos resultados sugerem que a hiperglicemia crônica do DM1 não interfere com
os mecanismos liberadores de GH, ACTH e cortisol estimulados por estes
Tabela 1 Dados clínicos do grupo controle.
Indiv. Sexo Idade (anos) IMC1(kg/m2)
1 M 28 23,3
2 M 30 22,4
3 M 30 25,5
4 F 34 20,9
5 F 35 23,7
6 F 20 24,3
7 M 26 24,9
8 M 28 25,9
9 M 25 18,8
Média 28,4 23,3
DP 4,6 2,3
EP 1,5 0,8
Tabela 2 Dados clínicos dos pacientes com diabetes tipo 1.
Indiv. Sexo Idade (anos) IMC1(kg/m2)
1 M 27 22,7
2 F 23 23,0
3 F 20 22,4
4 M 18 24,5
5 F 29 18,1
6 M 30 18,9
7 M 26 21,1
8 M 19 22,4
9 F 25 18,1
Média 24,1 21,2
DP 4,4 2,3
EP 1,5 0,8
Tabela 3 Valores individuais e níveis médios das dosagens basais do grupo controle. Indiv. GH (Qg/L) IGF 1 (ng/mL) ACTH (pg/mL) Cortisol (Qg/dL) Glicose (mg/dL) HbA1c (%)
1 0,1 154,0 27,6 12,0 84 4,8
2 0,1 164,0 7,7 10,1 87 5,2
3 0,2 132,0 8,1 8,7 84 5,0
4 0,1 126,0 14,4 7,4 74 5,2
5 0,2 228,0 8,5 4,7 83 4,6
6 0,3 323,0 9,6 7,5 84 5,0
7 0,1 180,0 22,2 8,4 88 5,3
8 2,1 142,0 16,6 5,1 77 4,8
9 1,9 165,0 15,5 9,8 89 5,0
Média 0,6 179,3 14,5 8,2 84 5,0
DP 0,8 61,8 6,9 2,3 6 0,2
EP 0,3 20,6 2,3 0,8 2 0,1
Tabela 4 Valores individuais e níveis médios das dosagens basais dos pacientes com diabetes tipo 1.
Indiv. GH (Qg/L) IGF 1 (ng/mL) ACTH (pg/mL) Cortisol (Qg/dL) Glicose (mg/dL) HbA1c (%)
1 11,3 99,6 28,4 18,0 167 8,3
2 5,5 159,0 9,6 13,7 208 14,7
3 1,9 168,0 7,3 9,2 159 21,4
4 3,4 169,0 15,6 5,9 324 10,4
5 4,2 215,0 20,1 12,3 187 9,0
6 3,6 125,0 29,9 13,3 208 12,9
7 0,3 152,0 35,1 13,7 97 8,0
8 0,1 220,0 23,4 15,0 266 10,6
9 1,0 184,0 9,4 4,1 179 9,6
Média 3,5 165,7 19,9 11,7 200 11,7
DP 3,5 38,7 10,1 4,5 65 4,0
Tabela 5 Valores individuais e níveis médios de GH após a administração de ghrelina em controles.
GH (Sg/L)
Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 0,04 34,6 87,8 101,9 56,7 30,7 22,6 10,7 6,5
2 0,1 33,2 37,9 29,0 21,5 15,6 9,8 6,0 4,2
3 0,5 17,1 20,9 16,2 13,3 9,5 6,8 2,4 1,7
4 0,1 17,0 40,0 34,4 23,7 10,7 8,1 4,9 2,8
5 0,07 15,4 24,0 13,3 6,8 3,9 2,1 1,2 0,7
6 0,13 118,5 191,0 165,3 117,0 72,8 33,9 18,8 14,2
7 0,3 27,9 37,4 31,1 22,3 14,0 8,3 5,0 2,9
8 0,9 34,5 35,8 32,0 25,8 17,4 16,4 12,0 7,9
9 0,8 16,5 29,9 23,6 16,3 11,1 7,4 4,9 3,8
Média 0,3 35,0 56,1 49,6 33,7 20,6 12,8 7,3 5,0
DP 0,3 32,4 54,2 50,7 34,2 20,9 9,9 5,5 4,1
EP 0,1 10,8 18,1 16,9 11,4 7,0 3,3 1,8 1,4
Tabela 6 Valores individuais e níveis médios de GH após a administração de GHRP"6 em controles.
GH (Sg/L)
Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 0,07 12,0 25,0 24,0 16,7 9,4 5,4 3,3 1,0
2 0,05 14,0 20,6 15,8 9,1 4,2 2,5 1,5 1,0
3 0,02 19,7 20,7 18,1 12,2 7,8 2,9 1,7 1,0
4 0,09 10,7 27,4 20,0 12,3 8,1 4,3 2,8 1,4
5 0,2 5,2 10,4 7,9 4,0 2,9 2,3 2,0 1,4
6 0,2 28,7 37,5 37,6 27,6 20,6 12,0 7,3 4,7
7 0,07 17,5 26,4 18,5 10,9 6,5 3,4 2,0 1,1
8 5,2 35,7 31,7 26,0 21,7 16,1 11,3 7,9 4,9
9 4,5 15,5 14,7 10,2 8,1 4,9 2,8 1,7 1,4
Média 1,2 17,7 23,8 19,8 13,6 8,9 5,2 3,4 2,0
DP 2,1 9,4 8,3 8,9 7,3 5,8 3,8 2,5 1,6
Tabela 7 Valores individuais e níveis médios de GH após a administração de GHRH em controles.
GH (Sg/L)
Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 0,08 8,0 8,7 7,4 7,4 6,7 5,5 3,8 2,2
2 0,1 5,9 3,9 3,3 3,1 2,6 2,1 1,2 1,0
3 0,03 4,2 5,1 5,9 4,6 3,2 2,2 1,3 0,9
4 0,2 6,9 7,6 6,3 4,7 3,2 2,0 0,6 0,5
5 0,2 14,4 19,3 23,3 20,9 19,2 12,8 15,3 12,8
6 0,6 7,1 7,1 10,4 9,9 8,8 11,4 7,0 3,9
7 0,02 0,03 5,9 6,3 5,8 4,1 2,4 1,3 0,9
8 0,2 11,6 7,8 4,6 3,7 2,8 1,8 1,6 1,4
9 0,4 3,3 2,4 1,7 1,4 1,5 1,8 1,3 0,9
Média 0,2 6,8 7,5 7,7 6,8 5,8 4,7 3,7 2,7
DP 0,2 4,3 4,9 6,4 5,8 5,5 4,4 4,8 3,9
EP 0,1 1,4 1,6 2,1 1,9 1,8 1,5 1,6 1,3
Tabela 8 Valores individuais e níveis médios de GH após a administração de ghrelina em pacientes com diabetes tipo 1.
GH (Sg/L) Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 11,1 72,0 55,6 34,3 22,4 14,1 8,9 5,3 4,0
2 7,9 121,6 82,0 53,6 22,5 15,3 9,2 7,5 4,4
3 0,2 41,6 55,2 30,2 14,1 9,8 4,4 2,9 1,7
4 1,0 80,0 80,0 84,0 33,0 24,1 17,3 16,0 7,0
5 2,2 16,4 27,2 26,0 13,5 12,0 6,0 3,6 2,7
6 6,9 72,8 89,8 58,8 22,7 14,9 7,9 5,0 3,7
7 0,2 42,0 50,0 55,6 35,1 24,0 16,0 16,3 7,5
8 0,04 28,0 75,6 114,8 42,8 25,6 19,6 12,1 7,1
9 1,1 38,8 50,8 36,0 10,2 6,2 2,5 2,9 2,1
Média 3,4 57,0 62,9 54,8 24,0 16,2 10,2 8,0 4,5
DP 4,1 32,5 20,1 28,9 11,0 6,9 6,0 5,4 2,2
Tabela 9 Valores individuais e níveis médios de GH após a administração de GHRP"6 em pacientes com diabetes tipo 1.
GH (Sg/L) Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 14,5 54,4 44,8 28,9 20,9 14,6 8,7 6,3 4,0
2 3,2 26,4 12,0 4,5 2,9 2,1 1,2 1,0 1,2
3 4,5 38,8 43,2 20,5 14,9 10,9 7,0 4,6 3,1
4 4,4 56,0 50,8 26,2 23,1 24,1 10,3 5,9 5,7
5 10,0 18,8 14,0 11,2 7,0 4,0 2,4 1,5 0,9
6 1,3 54,0 46,0 24,0 14,6 8,8 5,3 3,4 2,7
7 0,2 17,2 31,2 24,3 21,4 12,3 8,0 5,1 3,0
8 0,06 24,4 42,8 27,0 22,0 15,3 8,1 4,4 2,6
9 1,4 36,0 28,8 12,1 6,6 5,1 4,2 3,7 3,6
Média 4,4 36,2 34,8 19,9 14,8 10,8 6,1 4,0 3,0
DP 4,9 15,6 14,2 8,5 7,7 6,8 3,1 1,8 1,4
EP 1,6 5,2 4,7 2,8 2,6 2,3 1,0 0,6 0,5
Tabela 10 Valores individuais e níveis médios de GH após a administração de GHRH em pacientes com diabetes tipo 1.
GH (Sg/L) Tempo (min)
Indiv. 0 15 30 45 60 75 90 105 120
1 8,2 45,2 37,2 22,5 11,0 10,3 6,7 3,8 3,5
2 5,3 21,6 12,0 6,6 7,6 7,7 6,9 4,9 3,9
3 1,0 4,4 5,2 4,5 3,7 2,7 2,5 3,1 3,8
4 4,8 31,2 18,8 13,2 5,7 3,6 2,4 1,6 1,2
5 0,4 22,0 29,2 16,6 12,0 12,0 12,4 11,7 8,4
6 2,5 5,2 7,2 14,1 13,8 13,1 11,8 8,7 6,3
7 0,4 22,4 20,0 16,0 10,4 8,7 5,4 4,3 2,7
8 0,2 9,6 18,4 16,1 16,9 15,3 11,2 5,6 3,5
9 0,6 4,8 3,2 1,5 1,7 1,0 0,4 0,3 0,3
Média 2,6 18,5 16,8 12,3 9,2 8,3 6,6 4,9 3,7
DP 2,9 13,9 11,3 6,8 4,9 5,0 4,4 3,5 2,4
Tabela 11 Valores individuais e níveis médios de cortisol após a administração de ghrelina em controles.
Cortisol (Sg/dL)
Tempo (min)
Indiv. 0 15 30 45 60 90
1 9,6 12,2 15,2 14,9 14,1 13,6
2 7,3 10,2 16,4 18,1 17,9 12,7
3 9,2 15,2 17,7 20,1 19,6 17,3
4 9,6 10,6 12,5 10,2 9,4 8,5
5 6,3 8,6 10,7 9,3 7,6 7,5
6 7,5 21,5 18,8 17,7 16,7 14,8
7 6,9 11,3 14,9 14,2 11,4 8,4
8 5,3 9,1 11,0 9,4 11,2 7,8
9 10,1 15,2 19,6 18,0 16,0 13,9
Média 8,0 12,7 15,2 14,7 13,8 11,6
DP 1,7 4,1 3,3 4,2 4,1 3,6
EP 0,6 1,4 1,1 1,4 1,4 1,2
Tabela 12 Valores individuais e níveis médios de cortisol após a administração de GHRP"6 em controles.
Cortisol (Sg/dL)
Tempo (min)
Indiv. 0 15 30 45 60 90
1 14,3 15,6 15,7 16,8 13,6 13,0
2 12,9 12,5 11,6 10,6 9,6 7,5
3 8,2 12,6 11,7 10,3 9,5 8,0
4 5,1 10,3 8,8 6,6 5,9 6,9
5 3,0 9,0 7,1 6,3 7,7 7,8
6 7,4 11,2 12,9 15,3 12,0 9,1
7 9,8 12,7 11,4 10,4 9,0 7,8
8 4,9 15,6 12,2 11,5 9,0 7,2
9 9,4 12,2 12,8 12,8 11,1 9,5
Média 8,3 12,4 11,6 11,2 9,7 8,5
DP 3,7 2,2 2,5 3,5 2,3 1,9
Tabela 13 Valores individuais e níveis médios de cortisol após a administração de ghrelina em pacientes com diabetes tipo 1.
Cortisol (Sg/dL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 19,1 22,4 20,1 20,6 18,4 15,7
2 9,0 15,5 16,5 14,9 14,5 11,6
3 10,4 11,0 13,2 12,1 8,4 6,8
4 4,5 7,7 11,5 12,3 10,7 7,6
5 9,4 14,5 16,6 20,2 22,8 17,3
6 17,4 20,9 18,6 20,9 17,4 14,0
7 14,5 18,3 17,7 18,7 16,8 14,3
8 15,8 16,2 18,6 17,7 14,0 13,3
9 4,8 14,0 18,4 15,6 13,7 8,4
Média 11,7 15,6 16,8 17,0 15,2 12,1
DP 5,3 4,6 2,8 3,4 4,3 3,7
EP 1,8 1,5 0,9 1,1 1,4 1,2
Tabela 14 Valores individuais e níveis médios de cortisol após a administração de GHRP"6 em pacientes com diabetes tipo 1.
Cortisol (Sg/dL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 16,8 19,7 17,7 17,8 16,4 13,9
2 18,4 19,9 16,9 13,9 12,3 13,9
3 8,0 13,7 18,9 19,9 16,1 11,6
4 7,2 14,0 13,2 12,3 10,2 4,7
5 15,2 14,2 14,3 12,9 13,2 7,1
6 9,2 13,6 13,2 11,2 10,8 6,4
7 12,9 15,0 16,5 14,2 12,9 10,5
8 14,1 15,8 15,1 12,4 12,2 10,0
9 3,4 11,9 12,9 16,3 9,0 4,6
Média 11,7 15,3 15,4 14,5 12,6 9,2
DP 5,0 2,8 2,2 2,9 2,5 3,6
Tabela 15 Valores individuais e níveis médios de ACTH após a administração de ghrelina em controles.
ACTH (pg/mL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 33,4 67,1 58,9 47,4 38,1 25,4
2 5,0 21,4 32,4 25,1 16,5 5,0
3 9,5 60,0 57,5 33,4 26,3 13,5
4 15,7 21,9 19,6 16,5 12,8 11,9
5 10,0 19,2 21,4 16,2 14,0 11,0
6 11,1 30,3 28,3 21,5 16,5 9,6
7 23,6 82,6 68,6 51,6 34,7 20,5
8 15,0 112,0 74,2 48,5 30,5 14,2
9 16,9 50,9 43,9 33,4 22,7 14,5
Média 15,6 51,7 45,0 32,6 23,6 14,0
DP 8,5 32,1 20,6 13,9 9,3 6,0
EP 2,8 10,7 6,9 4,6 3,1 2,0
Tabela 16 Valores individuais e níveis médios de ACTH após a administração de GHRP"6 em controles.
ACTH (pg/mL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 21,8 27,3 22,0 18,9 17,1 14,9
2 10,4 10,8 8,5 7,0 7,8 7,6
3 6,7 13,6 12,8 10,4 7,7 6,5
4 13,1 21,0 16,5 14,0 13,2 19,0
5 7,0 20,0 15,0 14,5 16,4 19,0
6 8,1 24,0 18,3 15,0 13,0 11,0
7 20,8 28,9 22,6 19,0 16,8 14,6
8 18,1 87,1 56,1 34,1 21,7 12,5
9 14,1 23,3 22,2 17,2 16,6 13,5
Média 13,3 28,4 21,6 16,7 14,5 13,2
DP 5,8 22,8 13,8 7,6 4,6 4,4
Tabela 17 Valores individuais e níveis médios de ACTH após a administração de ghrelina em pacientes com diabetes tipo 1.
ACTH (pg/mL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 25,0 37,8 23,8 17,4 15,2 10,4
2 6,3 14,6 14,3 9,8 7,4 6,8
3 7,2 11,8 10,0 8,9 9,9 5,7
4 12,2 111,0 63,3 53,1 42,1 23,4
5 13,3 193,0 123,0 81,5 55,7 29,3
6 43,4 76,2 51,9 33,0 22,1 14,9
7 44,5 99,2 76,7 59,5 43,2 28,7
8 25,5 41,1 30,4 24,6 25,8 19,5
9 8,8 28,8 28,0 19,1 16,5 9,7
Média 20,7 68,2 46,8 34,1 26,4 16,5
DP 14,9 58,8 36,3 25,2 16,8 9,1
EP 5,0 19,6 12,1 8,4 5,6 3,0
Tabela 18 Valores individuais e níveis médios de ACTH após a administração de GHRP"6 em pacientes com diabetes tipo 1.
ACTH (pg/mL) Tempo (min)
Indiv. 0 15 30 45 60 90
1 31,8 39,0 23,9 17,7 12,9 9,1
2 12,9 14,0 12,1 10,7 10,5 7,8
3 7,4 43,9 25,5 19,6 15,4 10,3
4 19,0 76,9 47,3 36,5 30,7 18,3
5 26,8 27,4 22,9 17,6 15,4 11,0
6 16,4 28,8 19,8 11,8 16,7 14,8
7 25,7 43,0 31,3 34,0 28,8 17,9
8 21,3 26,8 24,0 21,0 19,1 19,6
9 9,9 22,6 20,1 14,5 13,1 10,1
Média 19,0 35,8 25,2 20,4 18,1 13,2
DP 8,2 18,3 9,8 9,1 7,1 4,5
Tabela 19 Valores basais e níveis médios de glicose em controles.
Glicose(mg/dL)
Indiv. Ghrelina GHRP 6 GHRH
1 81 88 83
2 84 88 89
3 82 85 ND
4 73 75 ND
5 84 82 ND
6 81 86 ND
7 92 85 87
8 79 81 72
9 89 88 90
Média 83 84 84
DP 6 4 7
EP 2 1 3
ND: Não disponível
Tabela 20 Valores basais e níveis médios de glicose em pacientes com diabetes tipo 1.
Glicose(mg/dL)
Indiv. Ghrelina GHRP 6 GHRH
1 208 133 161
2 222 189 213
3 240 90 148
4 306 354 312
5 84 171 307
6 178 167 279
7 95 91 106
8 292 360 145
9 179 139 218
Média 200 188 210
DP 77 101 76
Tabela 21 Resposta de GH expressa em pico e ASC após a administração de GHRH, GHRP"6 e ghrelina em controles.
Pico (Sg/L) ASC (Sg/L.120 min)
Indiv. GHRH GHRP 6 Ghrelina GHRH GHRP 6 Ghrelina
1 8,7 25,0 101,9 730 1445 5224
2 5,9 20,6 37,9 340 1023 2327
3 5,9 20,7 20,9 404 1254 1310
4 7,6 27,4 40,0 475 1295 2104
5 23,3 10,4 24,0 1976 533 1006
6 11,4 37,6 191,0 959 2606 10867
7 6,3 26,4 37,4 394 1287 2214
8 11,6 35,7 35,8 521 2332 2675
9 3,3 15,5 29,9 211 913 1680
Média 9,3 24,4 57,6 668 1410 3267
DP 5,9 8,8 55,4 538 662 3097
EP 2,0 2,9 18,5 179 221 1032
Tabela 22 Resposta de GH expressa em pico e ASC após a administração de GHRH, GHRP"6 e ghrelina em pacientes com diabetes tipo 1.
Pico (Sg/L) ASC (Sg/L.120 min)
Indiv. GHRH GHRP 6 Ghrelina GHRH GHRP 6 Ghrelina
1 45,2 54,4 72,0 2138 2818 3302
2 21,6 26,4 121,6 1079 785 4768
3 5,2 43,2 55,2 428 2156 2387
4 31,2 56,0 84,0 1193 3022 5076
5 29,2 18,8 27,2 1805 965 1607
6 14,1 54,0 89,8 1175 2372 4158
7 22,4 31,2 55,6 1331 1817 3643
8 18,4 42,8 114,8 1424 2180 4831
9 4,8 36,0 50,8 200 1485 2235
Média 21,3 40,3 74,6 1197 1955 3556
DP 12,9 13,2 31,0 605 769 1262
Tabela 23 Resposta de cortisol expressa em pico e ASC após a administração de GHRP"6 e ghrelina em controles.
Pico (Sg/dL) ASC (Sg/dL.90 min)
Indiv. GHRP 6 Ghrelina GHRP 6 Ghrelina
1 16,8 15,2 1330 1258
2 12,9 18,1 946 1319
3 12,6 20,1 914 1565
4 10,3 12,5 660 911
5 9,0 10,7 649 760
6 15,3 21,5 1053 1524
7 12,7 14,9 911 1040
8 15,6 11,2 937 851
9 12,8 19,6 1030 1436
Média 13,1 16,0 937 1185
DP 2,5 4,0 205 303
EP 0,8 1,3 68 101
Tabela 24 Resposta de cortisol expressa em pico e ASC após a administração de GHRP"6 e ghrelina em pacientes com diabetes tipo 1.
Pico (Sg/dL) ASC (Sg/dL.90 min)
Indiv. GHRP 6 Ghrelina GHRP 6 Ghrelina
1 19,7 22,4 1532 1739
2 19,9 16,5 1384 1271
3 19,9 13,2 1384 914
4 13,2 12,3 947 861
5 15,2 22,8 1139 1613
6 13,6 20,9 978 1638
7 16,5 18,3 1230 1522
8 15,8 18,6 1180 1421
9 16,3 18,4 914 1190
Média 16,7 18,2 1187 1352
DP 2,6 3,7 217 316
Tabela 26 Resposta de ACTH expressa em pico e ASC após a administração de GHRP"6 e ghrelina em pacientes com diabetes com tipo 1.
Pico (Sg/dL) ASC (Sg/dL.90 min)
Indiv. GHRP 6 Ghrelina GHRP 6 Ghrelina
1 39,0 37,8 1874 1871
2 14,0 14,6 1002 896
3 43,9 11,8 1892 823
4 76,9 111,0 3518 4801
5 27,4 193,0 1731 7755
6 28,8 76,2 1627 3463
7 43,0 99,2 2734 5267
8 26,8 41,1 1961 2506
9 22,6 28,8 1379 1721
Média 35,8 68,2 1969 3234
DP 18,3 58,8 746 2319
EP 6,1 19,6 249 773
Tabela 25 Resposta de ACTH expressa em pico e ASC após a administração de GHRP"6 e ghrelina em controles.
Pico (pg/mL) ASC (pg/mL.90min)
Indiv. GHRP 6 Ghrelina GHRP 6 Ghrelina
1 27,3 67,1 1795 4090
2 10,8 32,4 762 1667
3 13,6 60,0 873 3128
4 21,0 21,9 1453 1454
5 20,0 21,4 1449 1407
6 24,0 30,3 1378 1800
7 28,9 82,6 1811 4307
8 87,1 112,0 3471 4532
9 23,3 50,9 1622 2778
Média 28,4 53,2 1624 2796
DP 22,8 30,6 783 1279
Tabela 27 Resposta de GH expressa em ∆ASC após a administração de GHRH, GHRP"6 e ghrelina em controles.
∆ASC (Sg/L.120 min)
Indiv. GHRH GHRP 6 Ghrelina
1 720 1437 5219
2 329 1017 2315
3 401 1252 1250
4 451 1284 2092
5 1952 509 998
6 887 2582 10851
7 392 1278 2178
8 497 1708 2567
9 163 373 1584
Média 643 1271 3228
DP 535 651 3109
EP 178 217 1036
Tabela 28 Resposta de GH expressa em ∆ASC após a administração de GHRH, GHRP"6 e ghrelina em pacientes com diabetes tipo 1.
∆ASC (Sg/L.120 min)
Indiv. GHRH GHRP 6 Ghrelina
1 1154 1078 1970
2 443 401 3820
3 308 1616 2363
4 617 2494 4956
5 1757 "235 1343
6 875 2216 3330
7 1283 1793 3619
8 1400 2173 4826
9 128 1317 2103
Média 885 1428 3148
DP 551 897 1282
Tabela 29 Resposta de cortisol expressa em ∆ASC após a administração de GHRP"6 e ghrelina em controles.
∆ASC (Sg/dL.90 min)
Indiv. GHRP 6 Ghrelina
1 43 394
2 "215 662
3 176 737
4 201 47
5 379 193
6 387 849
7 28 419
8 496 374
9 184 527
Média 187 467
DP 218 257
EP 73 86
Tabela 30 Resposta de cortisol expressa em ∆ASC após a administração de GHRP"6 e ghrelina em pacientes com diabetes tipo 1.
TASC (Sg/dL.90 min)
Indiv. GHRP 6 Ghrelina
1 20 20
2 "272 461
3 664 "23
4 299 456
5 "230 767
6 150 72
7 69 217
8 "89 "2
9 608 758
Média 135 303
DP 335 318
Tabela 31 Resposta de ACTH expressa em ∆ASC após a administração de GHRP"6 e ghrelina em controles.
∆ASC (pg/mL.90 min) Indiv. GHRP 6 Ghrelina
1 "167 1084
2 "174 1217
3 270 2273
4 274 41
5 819 507
6 649 801
7 "62 2183
8 1842 3182
9 353 1257
Média 423 1394
DP 634 982
EP 211 327
Tabela 32 Resposta de ACTH expressa em ∆ASC após a administração de GHRP"6 e ghrelina em pacientes com diabetes com tipo 1.
TASC (Sg/dL.90 min) Indiv. GHRP 6 Ghrelina
1 "988 "380
2 "159 329
3 1226 175
4 1808 3703
5 "681 6558
6 151 "443
7 421 1262
8 44 211
9 488 929
Média 257 1372
DP 872 2312
