Inhibition by dexamethasone of arginine vasopressin and ACTH responses to insulin-induced hypoglycemia and cigarette smoking in normal men P. Chiodera and V. Coiro
University Clinics of Endocrinology1
Abstract. Glucocorticoids are known
and Internal Medicine2, School of Medicine,
to inhibit the AVP response to osmotic and hemodynamic stimuli in humans. In the present study, we examined whether the AVP response to other primary stimuli, such as insulin-induced hypoglycemia and cigarette smoking was sensitive to inhibition by dexamethasone. The plasma ACTH responses were also measured. Twenty-four normal men were randomly divided into 3 groups of 8 subjects. One group was tested with insulin (0.15 IU/kg in an iv bolus) and another group with cigarette smoking (2 non-filter cigarettes smoked in succession within 10 min) with and without pretreatment with dexamethasone (2 or 4 mg in an iv bolus 10 min before insulin or smoking). The third group was tested with dexamethasone alone (2 or 4 mg in an iv bolus). The administration of dexamethasone (2 or 4 mg) alone did not modify the circulating levels of AVP and ACTH at any time during the next hour. The hypoglycemic response to insulin was similar regardless of dexamethasone treatment. AVP rose sharply in response to both hypoglycemia and smoking, reaching mean peak levels at 45 and 30 min, respectively. After both stimuli, the mean peak levels of AVP were 2.25 times higher than baseline. Pretreatment with dexamethasone (2 or 4 mg) significantly decreased the AVP responses to both hypoglycemia and cigarette smoking. Following pretreatment with dexamethasone (2 or 4 mg) the AVP increments in response to hypoglycemia or cigarette smoking were only 1.7 times higher than baseline. The plasma concentrations of ACTH were strikingly increased by hypoglycemia. The mean peak level was 3.5 times higher than baseline and was reached at 45 min. Pretreatment with dexamethasone (2 or 4 mg) significantly reduced hypoglycemia-induced ACTH increase, with a mean peak response 2.8 times higher than baseline. Cigarette smoking significantly increased the plasma ACTH concentrations. The mean peak level was observed at 20 min and
was
to
1.3 times
smoking
University of Parma, Parma, Italy
higher than baseline. The ACTH response significant following pretreatment
was not
with dexamethasone (2 or 4 mg). These data show a partial inhibitory effect of dexamethasone on hypoglycemia\x=req-\ and cigarette smoking-stimulated AVP secretion. In addition, the data show suppression by dexamethasone of the ACTH response to cigarette smoking and confirm previous observations of a partial inhibition by glucocorticoids of hypoglycemia-induced ACTH release.
A variety of studies have provided evidence of an involvement of glucocorticoids in the control of vasopressin secretion (reviewed in 1). Experiments performed in rats showed that dexamethasone has different effects on the vasopressin responses to different stimuli. In fact, dexamethasone has been found unable to change the vasopressin response to angiotensin II infusion, whereas it increased the vasopressin response to tail shock or to polyethyl¬ ene
glycol injected intraperitoneally (producing a
decrease of intravascular volume) and it decreased the vasopressin response to hypertonic saline stim¬ ulation (1). On the other hand, studies carried out in humans have shown that plasma arginine vasopres¬ sin (AVP) responses to osmotic (2) and volumetric (1) stimuli are attenuated by pretreatment with glu¬ cocorticoids. There are other AVP-releasing stimuli, such as insulin-induced hypoglycemia or cigarette smok¬ ing, which are independent of osmotic and/or hemodynamic changes (3-5). At present, it is un¬ known whether glucocorticoids influence the AVP responses to hypoglycemia and/or cigarette smok-
ing. In order to answer this question, we measured the AVP responsiveness to insulin-induced hy¬ poglycemia and cigarette smoking in normal men, with and without pretreatment with dexame¬ thasone. In addition, the concurrent ACTH re¬ sponse to either hypoglycemia or cigarette smoking was measured.
Subjects and Methods Twenty-four normal male volunteers between the ages of 27 and 37 years, habitual smokers of 10-13 cigarettes/day, participated in this study after giving informed consent. All men were in good health and were within 10% of their ideal body weight. None of them were taking drugs for at least 3 weeks before the experimental day or were allowed to smoke for 12 h before the experiments. In order to test the influence of different doses of de¬ xamethasone on AVP secretion and to avoid an excessive number of experiments in the same subjects, men were randomly divided into three groups of 8. Each group was tested three times on three different days at an interval of at least seven days between tests. One group was submitted to insulin tolerance test (ITT), and another group was tested with cigarette smoking. Tests were performed with and without previous treat¬ ment with 2 mg dexamethasone. On a different occasion, 4 mg dexamethasone was used. A control test with normal saline alone and tests with dexamethasone alone (2 or 4 mg) were performed in the third group. All tests were carried out with subjects lying in the re¬ cumbent position and fasting from the previous evening. At 08.00 h of the experimental day an iv catheter was inserted into the antecubital vein and was kept patent by a slow saline (NaCl 0.9%) infusion. It was used for insulin and/or dexamethasone administration and blood sam¬
pling. Insulin tolerance test
experiments
ITT. Basal blood samples were taken 30 min and just before the iv bolus injection of insulin (0.15 IU/kg, Ac-
blood samples next hour.
were
taken
at
10-min intervals
during the
Dexamethasone plus cigarette smoking test. A similar proce¬ dure as in the cigarette smoking test was followed, except for the iv injection of a bolus of 2 mg dexamethasone at time -30. On a different occasion 4 mg dexamethasone was given. In the control cigarette smoking test normal saline was injected instead of dexamethasone.
Control
experiments
Dexamethasone test. After withdrawal of a basal blood sample, 2 mg dexamethasone was injected at time 0 as an iv bolus. Further blood samples were taken at 10-min intervals during the next hour. On a different occasion 4 mg dexamethasone was given. Saline test. This test was performed as the dexamethasone test, except for the administration of normal saline in¬ stead of dexamethasone. Systolic and diastolic blood pressure was measured by sphygmomanometry at each sampling time during tests. Samples from all tests were used for the evaluation of AVP, ACTH, osmolality, and hematocrit content. Blood glucose levels were measured in samples taken during the insulin tolerance tests. Blood for AVP and ACTH deter¬ minations was chilled to 4°C immediately and plasma was separated and stored at 20°C until assayed. AVP (6) and ACTH (7) were measured by RIA as pre¬ viously described. The intra-assay and inter-assay coeffi¬ cients of variation were 8 and 12.8% for AVP, and 5.8 and 9.8% for ACTH, respectively. The sensitivity of the RIA was 1.1 pmol/1 for AVP and 2.2 pmol/1 for ACTH. Osmolality was determined with an advanced osraometer (Osmette S, Secta s.r.l.), hematocrit was measured by Drummond microhematocrit (Drummond Scientific —
Co, Broomall, CA). Statistical analysis was performed with the Wilcoxon matched pair rank sum test, analysis of variance, and Dun¬ can's test for comparison of single time points of the curve, as appropriate. Simple correlations were deter¬ mined by regression analysis. Data are reported as mean ± sem.
trapid MC®,
Novo, Copenhagen). Additional specimens taken at 15-min intervals during the next hour. In the control ITT, normal saline was injected instead of dexamethasone. were
Dexamethasone plus ITT. This test was performed as the previously described ITT, except for the iv injection of a bolus of 2 mg dexamethasone at time -30. On a different occasion, 4 mg dexamethasone was given. In the control ITT, an equal volume of normal saline (NaCl 0.9%) was injected instead of dexamethasone.
Cigarette smoking experiments Cigarette smoking test. At time 0 subjects smoked two nonfilter cigarettes in succession within 10 min. Additional
Results Administration of 2 or 4 mg dexamethasone or normal saline was unable to change the circulating concentrations of AVP or ACTH within the next hour (Fig. 1). Insulin induced a typical hypoglycémie reaction characterized by visible sweating, tachycardia, and symptoms of neuroglucopenia in all subjects at about 30 min after the injection. Blood glucose con¬ centrations declined with a similar pattern regard¬ less of dexamethasone (2 or 4 mg) administration
Dexamethasone or
Saline
3-
r*-4—TT—if-4a
3
t
#^¡r 20-1 -
>"#îH"4H8H
oJ
=:? 0
W
20
55
40
35
60min.
Fig.
1. Plasma AVP and ACTH levels (mean ±sem) after iv in¬ jection of 2 mg dexamethasone (•.•), 4 mg dexame¬ thasone (•-.-.-.-•) or saline (•-•) in 8 normal men.
(Fig. 2 lower panel). Insulin-induced hypoglycemia produced a significant increase in plasma AVP con¬ centrations. The mean peak level was 2.25 times higher than baseline and was observed at 45 min (Fig. 2 upper panel). When the insulin tolerance test was repeated following pretreatment with 2 mg dexamethasone, the hypoglycemia-induced AVP increase was significantly lower than in the ITT (p
University Clinics of Endocrinology1
Abstract. Glucocorticoids are known
and Internal Medicine2, School of Medicine,
to inhibit the AVP response to osmotic and hemodynamic stimuli in humans. In the present study, we examined whether the AVP response to other primary stimuli, such as insulin-induced hypoglycemia and cigarette smoking was sensitive to inhibition by dexamethasone. The plasma ACTH responses were also measured. Twenty-four normal men were randomly divided into 3 groups of 8 subjects. One group was tested with insulin (0.15 IU/kg in an iv bolus) and another group with cigarette smoking (2 non-filter cigarettes smoked in succession within 10 min) with and without pretreatment with dexamethasone (2 or 4 mg in an iv bolus 10 min before insulin or smoking). The third group was tested with dexamethasone alone (2 or 4 mg in an iv bolus). The administration of dexamethasone (2 or 4 mg) alone did not modify the circulating levels of AVP and ACTH at any time during the next hour. The hypoglycemic response to insulin was similar regardless of dexamethasone treatment. AVP rose sharply in response to both hypoglycemia and smoking, reaching mean peak levels at 45 and 30 min, respectively. After both stimuli, the mean peak levels of AVP were 2.25 times higher than baseline. Pretreatment with dexamethasone (2 or 4 mg) significantly decreased the AVP responses to both hypoglycemia and cigarette smoking. Following pretreatment with dexamethasone (2 or 4 mg) the AVP increments in response to hypoglycemia or cigarette smoking were only 1.7 times higher than baseline. The plasma concentrations of ACTH were strikingly increased by hypoglycemia. The mean peak level was 3.5 times higher than baseline and was reached at 45 min. Pretreatment with dexamethasone (2 or 4 mg) significantly reduced hypoglycemia-induced ACTH increase, with a mean peak response 2.8 times higher than baseline. Cigarette smoking significantly increased the plasma ACTH concentrations. The mean peak level was observed at 20 min and
was
to
1.3 times
smoking
University of Parma, Parma, Italy
higher than baseline. The ACTH response significant following pretreatment
was not
with dexamethasone (2 or 4 mg). These data show a partial inhibitory effect of dexamethasone on hypoglycemia\x=req-\ and cigarette smoking-stimulated AVP secretion. In addition, the data show suppression by dexamethasone of the ACTH response to cigarette smoking and confirm previous observations of a partial inhibition by glucocorticoids of hypoglycemia-induced ACTH release.
A variety of studies have provided evidence of an involvement of glucocorticoids in the control of vasopressin secretion (reviewed in 1). Experiments performed in rats showed that dexamethasone has different effects on the vasopressin responses to different stimuli. In fact, dexamethasone has been found unable to change the vasopressin response to angiotensin II infusion, whereas it increased the vasopressin response to tail shock or to polyethyl¬ ene
glycol injected intraperitoneally (producing a
decrease of intravascular volume) and it decreased the vasopressin response to hypertonic saline stim¬ ulation (1). On the other hand, studies carried out in humans have shown that plasma arginine vasopres¬ sin (AVP) responses to osmotic (2) and volumetric (1) stimuli are attenuated by pretreatment with glu¬ cocorticoids. There are other AVP-releasing stimuli, such as insulin-induced hypoglycemia or cigarette smok¬ ing, which are independent of osmotic and/or hemodynamic changes (3-5). At present, it is un¬ known whether glucocorticoids influence the AVP responses to hypoglycemia and/or cigarette smok-
ing. In order to answer this question, we measured the AVP responsiveness to insulin-induced hy¬ poglycemia and cigarette smoking in normal men, with and without pretreatment with dexame¬ thasone. In addition, the concurrent ACTH re¬ sponse to either hypoglycemia or cigarette smoking was measured.
Subjects and Methods Twenty-four normal male volunteers between the ages of 27 and 37 years, habitual smokers of 10-13 cigarettes/day, participated in this study after giving informed consent. All men were in good health and were within 10% of their ideal body weight. None of them were taking drugs for at least 3 weeks before the experimental day or were allowed to smoke for 12 h before the experiments. In order to test the influence of different doses of de¬ xamethasone on AVP secretion and to avoid an excessive number of experiments in the same subjects, men were randomly divided into three groups of 8. Each group was tested three times on three different days at an interval of at least seven days between tests. One group was submitted to insulin tolerance test (ITT), and another group was tested with cigarette smoking. Tests were performed with and without previous treat¬ ment with 2 mg dexamethasone. On a different occasion, 4 mg dexamethasone was used. A control test with normal saline alone and tests with dexamethasone alone (2 or 4 mg) were performed in the third group. All tests were carried out with subjects lying in the re¬ cumbent position and fasting from the previous evening. At 08.00 h of the experimental day an iv catheter was inserted into the antecubital vein and was kept patent by a slow saline (NaCl 0.9%) infusion. It was used for insulin and/or dexamethasone administration and blood sam¬
pling. Insulin tolerance test
experiments
ITT. Basal blood samples were taken 30 min and just before the iv bolus injection of insulin (0.15 IU/kg, Ac-
blood samples next hour.
were
taken
at
10-min intervals
during the
Dexamethasone plus cigarette smoking test. A similar proce¬ dure as in the cigarette smoking test was followed, except for the iv injection of a bolus of 2 mg dexamethasone at time -30. On a different occasion 4 mg dexamethasone was given. In the control cigarette smoking test normal saline was injected instead of dexamethasone.
Control
experiments
Dexamethasone test. After withdrawal of a basal blood sample, 2 mg dexamethasone was injected at time 0 as an iv bolus. Further blood samples were taken at 10-min intervals during the next hour. On a different occasion 4 mg dexamethasone was given. Saline test. This test was performed as the dexamethasone test, except for the administration of normal saline in¬ stead of dexamethasone. Systolic and diastolic blood pressure was measured by sphygmomanometry at each sampling time during tests. Samples from all tests were used for the evaluation of AVP, ACTH, osmolality, and hematocrit content. Blood glucose levels were measured in samples taken during the insulin tolerance tests. Blood for AVP and ACTH deter¬ minations was chilled to 4°C immediately and plasma was separated and stored at 20°C until assayed. AVP (6) and ACTH (7) were measured by RIA as pre¬ viously described. The intra-assay and inter-assay coeffi¬ cients of variation were 8 and 12.8% for AVP, and 5.8 and 9.8% for ACTH, respectively. The sensitivity of the RIA was 1.1 pmol/1 for AVP and 2.2 pmol/1 for ACTH. Osmolality was determined with an advanced osraometer (Osmette S, Secta s.r.l.), hematocrit was measured by Drummond microhematocrit (Drummond Scientific —
Co, Broomall, CA). Statistical analysis was performed with the Wilcoxon matched pair rank sum test, analysis of variance, and Dun¬ can's test for comparison of single time points of the curve, as appropriate. Simple correlations were deter¬ mined by regression analysis. Data are reported as mean ± sem.
trapid MC®,
Novo, Copenhagen). Additional specimens taken at 15-min intervals during the next hour. In the control ITT, normal saline was injected instead of dexamethasone. were
Dexamethasone plus ITT. This test was performed as the previously described ITT, except for the iv injection of a bolus of 2 mg dexamethasone at time -30. On a different occasion, 4 mg dexamethasone was given. In the control ITT, an equal volume of normal saline (NaCl 0.9%) was injected instead of dexamethasone.
Cigarette smoking experiments Cigarette smoking test. At time 0 subjects smoked two nonfilter cigarettes in succession within 10 min. Additional
Results Administration of 2 or 4 mg dexamethasone or normal saline was unable to change the circulating concentrations of AVP or ACTH within the next hour (Fig. 1). Insulin induced a typical hypoglycémie reaction characterized by visible sweating, tachycardia, and symptoms of neuroglucopenia in all subjects at about 30 min after the injection. Blood glucose con¬ centrations declined with a similar pattern regard¬ less of dexamethasone (2 or 4 mg) administration
Dexamethasone or
Saline
3-
r*-4—TT—if-4a
3
t
#^¡r 20-1 -
>"#îH"4H8H
oJ
=:? 0
W
20
55
40
35
60min.
Fig.
1. Plasma AVP and ACTH levels (mean ±sem) after iv in¬ jection of 2 mg dexamethasone (•.•), 4 mg dexame¬ thasone (•-.-.-.-•) or saline (•-•) in 8 normal men.
(Fig. 2 lower panel). Insulin-induced hypoglycemia produced a significant increase in plasma AVP con¬ centrations. The mean peak level was 2.25 times higher than baseline and was observed at 45 min (Fig. 2 upper panel). When the insulin tolerance test was repeated following pretreatment with 2 mg dexamethasone, the hypoglycemia-induced AVP increase was significantly lower than in the ITT (p
