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ACTH and corticosterone secretion following 2-deoxyglucose administration in intact and in hypothalamic deafferentated male rats
Brain Research, 305 (1984) 109-113 Elsevier
109
BRE 10160
ACTH and Corticosterone Secretion following 2-Deoxyglucose Administration in Intact and in Hypothalamic Deafferentated Male Rats J. WEIDENFELD, R. A. SIEGEL, A. P. CORCOS, V. HELED, N. CONFORTI and I. CHOWERS
Laboratories of Experimental Endocrinology and Neurophysiology, Department of Neurology, Hadassah University Hospital and Department of Medicine, Bikur Cholim Hospital affiliated with the Hebrew University-Hadassah Medical School Jerusalem (Israel) (Accepted December 13th, 1983)
Key words: 2-deoxyglucose- - ACTH - - corticosterone - - glucose - - hypothalamus
Adult male rats were given a single i.p. injection of 2-deoxyglucose (2-DG, 100-400 mg/kg b.wt). The animals were decapitated 1-4 h later and trunk blood was collected for ACTH, corticosterone (CS) and glucose determinations. Serum ACTH and CS were markedly elevated when compared with saline-treated animals; these elevations were correlated with given doses of 2-DG so that with the higher dose, an approximately 6-fold increase in serum levels of both hormones was observed. Injection of 2-DG up to 200 mg/kg did not change serum glucose levels; injection of 400 mg/kg of 2-DG increased serum glucose by approximately 2-fold. A time course study showed that levels of serum ACTH, CS and glucose were maximal 1 h after 2-DG administration and returned to basal values 3 h later. Injection of 2-DG to animals with complete hypothalamic deafferentation failed to induce any change in serum ACTH, CS or glucose. This study demonstrates that: (1) 2-DG can stimulate the hypothalamo-hypophyseal-adrenal (HHA) axis as measured by ACTH and CS; this effect is not related to blood glucose levels; (2) the HHA response to 2-DG is mediated by sites outside the mediobasal hypothalamus. INTRODUCTION The glucose analogue 2-deoxyglucose (2-DG) can influence various central nervous system (CNS) and peripheral functions as manifested by hyperglycemia6, hypothermia and modified eating behavior1, 9. As was previously shown, these actions of 2 - D G may be associated with modified endocrine functionsT,10. In fact, a few reports showed that administration of 2-DG can markedly increase corticosterone (CS) secretion 2,10. The reason for this adrenocortical hypersecretion is not yet clear. Studies on animals with hypothalamic deafferentated rats have suggested that the effect of 2-DG on adrenocortical secretion is mediated, at least in part, by the CNS 10. It is well established now that blood CS concentrations are not an adequate index of hypothalamo-hypophyseal-adrenal ( H H A ) activity. This notion is based on: (1) direct neural regulation of the adrenal function, (2) varying
adrenal sensitivity to A C T H , (3) binding and metabolism of CS, and (4) non-linearity of serum A C T H - C S relationship3,S,IL All previous studies regarding 2-DG effects on the H H A axis were based mainly on CS as an index of adrenocortical activity. It is therefore essential to measure serum A C T H directly in order to elucidate the mode of action of 2-DG on this axis. In the present study we have determined the temporal and dose-response relationship changes in serum A C T H and CS following administration of 2 - D G to male rats. To determine the involvement of CNS in the effects of 2 - D G upon the H H A axis, we have also measured the secretory responses of serum A C T H and CS to 2 - D G in rats with complete hypothalamic deafferentation (CHD). In addition, the relations between A C T H and CS changes and blood glucose changes following 2 - D G were investigated in intact and C H D rats.
Correspondence: J. Weidenfeld, Laboratory of Experimental Endocrinology, Department of Neurology, Hadassah University Hospital, Ein Kerem, Jerusalem, Israel. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
110 MATERIALSAND METHODS The experiments were carried out on male rats of the Hebrew University strain, weighing approximately 240 g. They were housed in the animal room of our laboratory in groups of 5-6 animals per cage, under artificial illumination between 06.00 and 18.00 h. Purina Chow and water were available ad libitum Ambient temperature was 22-23 °C. Complete hypothalamic deafferentation was performed according to the method of Halasz and Gorski 5, with minor modifications. The dimensions of the knife were: radius, 1.4 mm; height, 2.4 mm. In this preparation the medial basal hypothalamus (MBH) is completely isolated from the remainder of the CNS. This is achieved by joining the anterior and posterior deafferentations with two cuts in the rostral-caudal direction. The location of these longitudinal cuts is 1.4 mm lateral to the midline. Functional integrity of the MBH was verified 1 week postoperatively by examining the plasma CS response to ether stress 4. Only animals which responded adequately were used in the subsequent experiments. The experiments were performed 10 days postoperatively. One day prior to the experiment the rats were transferred to individual cages. All experiments were performed between 09.00 and 11.00 h. 2-DG, 100-400 mg/kg b.wt. i.p. in 0.5 ml saline was injected. Control animals received vehicle only. The animals were killed by decapitation 1 or 4 h after 2-DG administration. Upon decapitation trunk blood was collected for serum glucose, ACTH and CS determinations and brains were removed and fixed in formalin for verification of accuracy and completeness of the deafferentations. The brains were first examined macroscopically (Fig. 1). They were subsequently sliced in a coronal plane on a cryostatemounted microtome and examined histologically. The sections were not stained. This enabled us to determine whether the cuts were correctly positioned, and whether they extended down to the base of the brain, so that residual neural ties did not remain. Serum glucose measurements were performed using the glucose oxidase method. Serum ACTH was estimated by R I A using material supplied by CIS (France). The antiserum used was rabbit antiporcine ACTH, the reference preparation was synthetic hu-
Fig. 1. Rat brain viewed from the ventral aspect, demonstrating typical complete hypothalamic deafferentation. For the purpose of illustration approximately0.5 mm of tissue has been removed from the ventral surface in the horizontal plane.
man ACTHl_39, and the tracer was 125I-labelled porcine ACTH. Bound and free fractions were separated with charcoal (Norit GSX Activated). The limit of sensitivitiy of this R I A was 30 pg/ml, and the intraand interassay coefficients of variation were 9% and 18%, respectively. Recovery and parallelism with standard tests both produced correlation coefficents greater than 0.99. Serum CS levels were determined using a corticosterone-binding globulin (CBG) assay. The CBG source was plasma drawn from a male dog which had received 7 daily injections of estradiol benzoate (10 rag). The label was [1,2,6,7-3H]-corticosterone (New England Nuclear, Boston MA). CS was extracted with ethyl acetate. Incubations were carried out at 45 °C (for 5 min), after which a charcoal step achieved separation of bound and free fractions. The sensitivity limit of this assay was approximately 0.5 #g/100 ml. RESULTS Fig. 2 illustrates the effect of increasing dosage of
111
200!
E
I00 O
~
2DG
_ .0SALINE
I
I
2DG
30
zL
I
ll/
20
U
10
___ ~ ~ . I
DISCUSSION
......... -eSALINE
l
-g
turned to baseline levels. In order to investigate the site of action of 2-DG in stimulating the H H A axis, we utilized rats with complete hypothalamic deafferentation. Fig. 4 shows that in intact animals, 2-DG raised the circulating levels of CS, A C T H and glucose as expected. On the contrary in C H D animals these responses were completely inhibited, so that no increases whatsoever were apparent.
l
150
,,,J
~L
/
2DG
The present experiments demonstrate that systemic acute administration of 2-DG leads to elevated serum CS and A C T H . These results support and extend previous studies 2,t° by providing direct evidence that serum A C T H concentration is also increased following injection of 2-DG. In view of the suggested
O~ -r
75
xx
200 =
U
.......
0
• SALINE
i
I
J
ioo
200
4O0
2 DG rnglkg
ID
100
0 U
Fig. 2. Effect of saline or increasing dosage of 2-deoxyglucose (2-DG) on circulating blood levels of ACTH, CS and glucose. The animals were sacrificed 1 h after 2-DG injection. Significance levels refer to comparison with saline treated group according to Student's t-test. *P < 0.001; **P < 0.01. Number of animals, 10-15 per group.
SALINE
3 0
I
I
I
I
X
30 .-e
2o
E~
2-DG on circulating levels of A C T H , CS and glucose. Blood samples were collected 1 h after injection of 2-DG. As little as 200 mg of 2-DG stimulated the secretion of both CS and A C T H ; this response was dose-dependent, as witnessed by the additional increase in circulating levels of both hormones, following administration of 400 mg of 2-DG. On the other hand, 200 mg of 2-DG had no effect upon blood glucose; the largest dose, however, 400 mg led to marked hyperglycemia, with blood glucose levels reaching approximately 200 mg%. The temporal aspects of CS, A C T H and glucose responses to 2-DG are illustrated in Fig. 3. The dose of 2-DG tested was 400 mg/kg. At 1 h post-injection, circulating levels of both hormones as well as glucose were markedly elevated, while after 4 h these had re-
=k
m u
20G
10 ....
-It-
~- ....
C .... i ..... ?
100
sA~,NE
20G
I-U
.... ~ ...............
~
SALINE
0 0
I
time
2
3
4
hours
Fig. 3. Effect of saline or 2-deoxyglucose (2-DG, 400 mg/kg b.wt.) on circulating blood levels of A C T H , CS and glucose as a function of time following 2-DG injection. For legend details see Fig. 1.
112
loo
N
CHO
u
o N
30 20
I0
CHD
2HD
N
R' T
b
40L It o
Saline
2DG
40Omg/Wg CHO • COMPLETE HYPOTHALAMIC DEAFF£RENTATION. M- INTACT
Fig. 4. Serum ACTH, CS and blood glucose in intact rats and in rats with complete hypothalamic deafferentation (CHD), 1 h after injection of either saline or 400 mg/kg b.wt. of 2-deoxyglucose (2-DG). Number of animals, 8-10 per group. Significance levels refer to comparison with 2-DG-treated CHD group with saline-treated N or CHD groups. *P < 0.001.
non-humoral regulation of adrenocortical function3,8, it could be assumed that the increased circulating CS level resulted from a direct neural activation (not ACTH mediated) of the adrenal cortex. The data of this study, showing clear temporal and dose response changes in serum ACTH, parallel to those of CS, (Figs. 2 and 3) suggest that the CS response was due to increased ACTH release. Our experiment on rats with CHD showed that responses in serum ACTH and CS to high dosage of 2-DG in these animals were completely abolished. These results clearly indicate that intact neuronal connections between the MBH and extra-MBH brain regions are essential for adrenocortical response to 2-DG. A previous report however, by Sun
et al. 1°, demonstrated that complete hypothalamic deafferentation only attenuated the response in serum CS to 2-DG by approximately 30%. This discrepancy, between our data and theirs, may be due to several methodological differences, namely: differences in Halasz knife dimensions (1.4 mm vs 1.2 mm), or differences in time span between the brain operations and 2-DG injections. It is of interest to note that we have previously shown n that in rats with CHD, operated in our laboratory, enhanced insulininduced hypoglycemia could elicit full adrenocortical response. This observation and the present data suggest that the activation of H H A axis following 2-DG and the response during enhanced hypoglycemia depends on two different mechanisms. The response to 2-DG is probably mediated by extra-MBH sites which are sensitive to impaired intracellular glucose utilization, whereas the response to deep hypoglycemia is mediated by sites located inside the MBH. Experiments to characterize the neurochemical nature of these mechanisms are carried out at present in our laboratory. Administration of 2-DG is known to induce hyperglycemia probably by an effect on the CNS 6. Since 2-DG also stimulates glucocorticoids secretion, its hyperglycemic effect could be related at least in part to the increased secretion of adrenocortical hormones. The present data showed that the time course changes in CS levels following 2-DG were similar to those of blood glucose. In addition, the lowest 2-DG dose which significantly increased serum CS, failed to alter blood glucose levels. These findings suggest that the 2-DG-induced hyperglycemia is not mediated by the activation of the adrenocortical function. The present experiments also demonstrated that in CHD animals no elevations in blood glucose following 2-DG could be detected. This finding supports a previous study showing a central site for the hyperglycemic action of 2-DG 6 and also indicates that this action is mediated by extra-MBH brain regions. In summary, the present study indicates that the CS secretory response to 2-DG is mediated by adenohypophyseal ACTH and that 2-DG induces hyperadrenocortical function and hyperglycemia by acting upon CNS sites located outside the MBH region.
113 REFERENCES 1 Berthoud, H. R. and Baetting, K., Effect of insulin and 2-deoxy-D-glucose on plasma glucose level an lateral hypothalamic eating threshold in the rat, Physiol. Behav., 12 (1973) 547-556. 2 Chevrier, C., Hammiche, N. and Brudieux, R., Effects of 2-deoxy-D-glucose on plasma and adrenal concentrations of corticosterone and aldosterone in standard diet fed and potassium loaded rats, Horm. Metabol. Res., 14 (1982) 94-97. 3 Dallman, M. F. and Yates, F. E., Dynamic asymmetries in the corticosteroid feedback pattern and distribution-metabolism-binding elements of adrenocortical system, Ann. N.Y. Acad. Sci., 156 (1969) 696-721. 4 Feldman, S., Conforti, N., Chowers, I. and Davidson, J. M., Differential effects of hypothalamic deafferentation on responses to different stresses, lsr. J. reed. Sci., 4 (1968) 908-911. 5 Halasz, B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrinology, 80 (1967) 608-622. 6 Muller, E. E., Cocchi, D. and Forni, A., A central site for the hyperglycemic action of 2-deoxy-D-glucose in mouse
and rat, Life Sci., 10 (1971) 1057-1067. 7 Roth, J., Glick, S. M., Yalow, R. S. and Berson, S. A., The influence of blood glucose on plasma concentration of growth hormone, Diabetes, 13 (1962) 355-361. 8 Siegel, R., Chowers, I., Conforti, N. and Feldman, S., Corticotrophin and corticosterone secretory patterns following acute neurogenic stress, in intact and in variously hypothalamic deafferentated male rats, Brain Research, 188 (1980) 399-410. 9 Smith, G. P. and Epstein, A. N., Increased feeding in response to decreased glucose utilization in the rat and monkey, Amer. J. Physiol., 217 (1969) 1083-1087. 10 Sun, C. L., Thoa, N. B. and Kopin, I. J., Comparison of the effects of 2-deoxyglucose and immobilization on plasma levels of catecholamines and corticosterone in awake rats, Endocrinology, 105 (1979) 306-310. 11 Weidenfeld, J., Siegel, R. A., Feldman, S., Conforti, N. and Chowers, I., ACTH and corticosterone secretion following insulin in intact and in variously hypothalamic deafferentated male rats, Exp. Brain Res., 48 (1982) 306-308. 12 Yasuda, N., Takebe, K. and Greer, M. A., Evidence of nycterohemeral period in stress-induced pituitary-adrenal activation, Neuroendocrinology, 21 (1976) 214-224.
109
BRE 10160
ACTH and Corticosterone Secretion following 2-Deoxyglucose Administration in Intact and in Hypothalamic Deafferentated Male Rats J. WEIDENFELD, R. A. SIEGEL, A. P. CORCOS, V. HELED, N. CONFORTI and I. CHOWERS
Laboratories of Experimental Endocrinology and Neurophysiology, Department of Neurology, Hadassah University Hospital and Department of Medicine, Bikur Cholim Hospital affiliated with the Hebrew University-Hadassah Medical School Jerusalem (Israel) (Accepted December 13th, 1983)
Key words: 2-deoxyglucose- - ACTH - - corticosterone - - glucose - - hypothalamus
Adult male rats were given a single i.p. injection of 2-deoxyglucose (2-DG, 100-400 mg/kg b.wt). The animals were decapitated 1-4 h later and trunk blood was collected for ACTH, corticosterone (CS) and glucose determinations. Serum ACTH and CS were markedly elevated when compared with saline-treated animals; these elevations were correlated with given doses of 2-DG so that with the higher dose, an approximately 6-fold increase in serum levels of both hormones was observed. Injection of 2-DG up to 200 mg/kg did not change serum glucose levels; injection of 400 mg/kg of 2-DG increased serum glucose by approximately 2-fold. A time course study showed that levels of serum ACTH, CS and glucose were maximal 1 h after 2-DG administration and returned to basal values 3 h later. Injection of 2-DG to animals with complete hypothalamic deafferentation failed to induce any change in serum ACTH, CS or glucose. This study demonstrates that: (1) 2-DG can stimulate the hypothalamo-hypophyseal-adrenal (HHA) axis as measured by ACTH and CS; this effect is not related to blood glucose levels; (2) the HHA response to 2-DG is mediated by sites outside the mediobasal hypothalamus. INTRODUCTION The glucose analogue 2-deoxyglucose (2-DG) can influence various central nervous system (CNS) and peripheral functions as manifested by hyperglycemia6, hypothermia and modified eating behavior1, 9. As was previously shown, these actions of 2 - D G may be associated with modified endocrine functionsT,10. In fact, a few reports showed that administration of 2-DG can markedly increase corticosterone (CS) secretion 2,10. The reason for this adrenocortical hypersecretion is not yet clear. Studies on animals with hypothalamic deafferentated rats have suggested that the effect of 2-DG on adrenocortical secretion is mediated, at least in part, by the CNS 10. It is well established now that blood CS concentrations are not an adequate index of hypothalamo-hypophyseal-adrenal ( H H A ) activity. This notion is based on: (1) direct neural regulation of the adrenal function, (2) varying
adrenal sensitivity to A C T H , (3) binding and metabolism of CS, and (4) non-linearity of serum A C T H - C S relationship3,S,IL All previous studies regarding 2-DG effects on the H H A axis were based mainly on CS as an index of adrenocortical activity. It is therefore essential to measure serum A C T H directly in order to elucidate the mode of action of 2-DG on this axis. In the present study we have determined the temporal and dose-response relationship changes in serum A C T H and CS following administration of 2 - D G to male rats. To determine the involvement of CNS in the effects of 2 - D G upon the H H A axis, we have also measured the secretory responses of serum A C T H and CS to 2 - D G in rats with complete hypothalamic deafferentation (CHD). In addition, the relations between A C T H and CS changes and blood glucose changes following 2 - D G were investigated in intact and C H D rats.
Correspondence: J. Weidenfeld, Laboratory of Experimental Endocrinology, Department of Neurology, Hadassah University Hospital, Ein Kerem, Jerusalem, Israel. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
110 MATERIALSAND METHODS The experiments were carried out on male rats of the Hebrew University strain, weighing approximately 240 g. They were housed in the animal room of our laboratory in groups of 5-6 animals per cage, under artificial illumination between 06.00 and 18.00 h. Purina Chow and water were available ad libitum Ambient temperature was 22-23 °C. Complete hypothalamic deafferentation was performed according to the method of Halasz and Gorski 5, with minor modifications. The dimensions of the knife were: radius, 1.4 mm; height, 2.4 mm. In this preparation the medial basal hypothalamus (MBH) is completely isolated from the remainder of the CNS. This is achieved by joining the anterior and posterior deafferentations with two cuts in the rostral-caudal direction. The location of these longitudinal cuts is 1.4 mm lateral to the midline. Functional integrity of the MBH was verified 1 week postoperatively by examining the plasma CS response to ether stress 4. Only animals which responded adequately were used in the subsequent experiments. The experiments were performed 10 days postoperatively. One day prior to the experiment the rats were transferred to individual cages. All experiments were performed between 09.00 and 11.00 h. 2-DG, 100-400 mg/kg b.wt. i.p. in 0.5 ml saline was injected. Control animals received vehicle only. The animals were killed by decapitation 1 or 4 h after 2-DG administration. Upon decapitation trunk blood was collected for serum glucose, ACTH and CS determinations and brains were removed and fixed in formalin for verification of accuracy and completeness of the deafferentations. The brains were first examined macroscopically (Fig. 1). They were subsequently sliced in a coronal plane on a cryostatemounted microtome and examined histologically. The sections were not stained. This enabled us to determine whether the cuts were correctly positioned, and whether they extended down to the base of the brain, so that residual neural ties did not remain. Serum glucose measurements were performed using the glucose oxidase method. Serum ACTH was estimated by R I A using material supplied by CIS (France). The antiserum used was rabbit antiporcine ACTH, the reference preparation was synthetic hu-
Fig. 1. Rat brain viewed from the ventral aspect, demonstrating typical complete hypothalamic deafferentation. For the purpose of illustration approximately0.5 mm of tissue has been removed from the ventral surface in the horizontal plane.
man ACTHl_39, and the tracer was 125I-labelled porcine ACTH. Bound and free fractions were separated with charcoal (Norit GSX Activated). The limit of sensitivitiy of this R I A was 30 pg/ml, and the intraand interassay coefficients of variation were 9% and 18%, respectively. Recovery and parallelism with standard tests both produced correlation coefficents greater than 0.99. Serum CS levels were determined using a corticosterone-binding globulin (CBG) assay. The CBG source was plasma drawn from a male dog which had received 7 daily injections of estradiol benzoate (10 rag). The label was [1,2,6,7-3H]-corticosterone (New England Nuclear, Boston MA). CS was extracted with ethyl acetate. Incubations were carried out at 45 °C (for 5 min), after which a charcoal step achieved separation of bound and free fractions. The sensitivity limit of this assay was approximately 0.5 #g/100 ml. RESULTS Fig. 2 illustrates the effect of increasing dosage of
111
200!
E
I00 O
~
2DG
_ .0SALINE
I
I
2DG
30
zL
I
ll/
20
U
10
___ ~ ~ . I
DISCUSSION
......... -eSALINE
l
-g
turned to baseline levels. In order to investigate the site of action of 2-DG in stimulating the H H A axis, we utilized rats with complete hypothalamic deafferentation. Fig. 4 shows that in intact animals, 2-DG raised the circulating levels of CS, A C T H and glucose as expected. On the contrary in C H D animals these responses were completely inhibited, so that no increases whatsoever were apparent.
l
150
,,,J
~L
/
2DG
The present experiments demonstrate that systemic acute administration of 2-DG leads to elevated serum CS and A C T H . These results support and extend previous studies 2,t° by providing direct evidence that serum A C T H concentration is also increased following injection of 2-DG. In view of the suggested
O~ -r
75
xx
200 =
U
.......
0
• SALINE
i
I
J
ioo
200
4O0
2 DG rnglkg
ID
100
0 U
Fig. 2. Effect of saline or increasing dosage of 2-deoxyglucose (2-DG) on circulating blood levels of ACTH, CS and glucose. The animals were sacrificed 1 h after 2-DG injection. Significance levels refer to comparison with saline treated group according to Student's t-test. *P < 0.001; **P < 0.01. Number of animals, 10-15 per group.
SALINE
3 0
I
I
I
I
X
30 .-e
2o
E~
2-DG on circulating levels of A C T H , CS and glucose. Blood samples were collected 1 h after injection of 2-DG. As little as 200 mg of 2-DG stimulated the secretion of both CS and A C T H ; this response was dose-dependent, as witnessed by the additional increase in circulating levels of both hormones, following administration of 400 mg of 2-DG. On the other hand, 200 mg of 2-DG had no effect upon blood glucose; the largest dose, however, 400 mg led to marked hyperglycemia, with blood glucose levels reaching approximately 200 mg%. The temporal aspects of CS, A C T H and glucose responses to 2-DG are illustrated in Fig. 3. The dose of 2-DG tested was 400 mg/kg. At 1 h post-injection, circulating levels of both hormones as well as glucose were markedly elevated, while after 4 h these had re-
=k
m u
20G
10 ....
-It-
~- ....
C .... i ..... ?
100
sA~,NE
20G
I-U
.... ~ ...............
~
SALINE
0 0
I
time
2
3
4
hours
Fig. 3. Effect of saline or 2-deoxyglucose (2-DG, 400 mg/kg b.wt.) on circulating blood levels of A C T H , CS and glucose as a function of time following 2-DG injection. For legend details see Fig. 1.
112
loo
N
CHO
u
o N
30 20
I0
CHD
2HD
N
R' T
b
40L It o
Saline
2DG
40Omg/Wg CHO • COMPLETE HYPOTHALAMIC DEAFF£RENTATION. M- INTACT
Fig. 4. Serum ACTH, CS and blood glucose in intact rats and in rats with complete hypothalamic deafferentation (CHD), 1 h after injection of either saline or 400 mg/kg b.wt. of 2-deoxyglucose (2-DG). Number of animals, 8-10 per group. Significance levels refer to comparison with 2-DG-treated CHD group with saline-treated N or CHD groups. *P < 0.001.
non-humoral regulation of adrenocortical function3,8, it could be assumed that the increased circulating CS level resulted from a direct neural activation (not ACTH mediated) of the adrenal cortex. The data of this study, showing clear temporal and dose response changes in serum ACTH, parallel to those of CS, (Figs. 2 and 3) suggest that the CS response was due to increased ACTH release. Our experiment on rats with CHD showed that responses in serum ACTH and CS to high dosage of 2-DG in these animals were completely abolished. These results clearly indicate that intact neuronal connections between the MBH and extra-MBH brain regions are essential for adrenocortical response to 2-DG. A previous report however, by Sun
et al. 1°, demonstrated that complete hypothalamic deafferentation only attenuated the response in serum CS to 2-DG by approximately 30%. This discrepancy, between our data and theirs, may be due to several methodological differences, namely: differences in Halasz knife dimensions (1.4 mm vs 1.2 mm), or differences in time span between the brain operations and 2-DG injections. It is of interest to note that we have previously shown n that in rats with CHD, operated in our laboratory, enhanced insulininduced hypoglycemia could elicit full adrenocortical response. This observation and the present data suggest that the activation of H H A axis following 2-DG and the response during enhanced hypoglycemia depends on two different mechanisms. The response to 2-DG is probably mediated by extra-MBH sites which are sensitive to impaired intracellular glucose utilization, whereas the response to deep hypoglycemia is mediated by sites located inside the MBH. Experiments to characterize the neurochemical nature of these mechanisms are carried out at present in our laboratory. Administration of 2-DG is known to induce hyperglycemia probably by an effect on the CNS 6. Since 2-DG also stimulates glucocorticoids secretion, its hyperglycemic effect could be related at least in part to the increased secretion of adrenocortical hormones. The present data showed that the time course changes in CS levels following 2-DG were similar to those of blood glucose. In addition, the lowest 2-DG dose which significantly increased serum CS, failed to alter blood glucose levels. These findings suggest that the 2-DG-induced hyperglycemia is not mediated by the activation of the adrenocortical function. The present experiments also demonstrated that in CHD animals no elevations in blood glucose following 2-DG could be detected. This finding supports a previous study showing a central site for the hyperglycemic action of 2-DG 6 and also indicates that this action is mediated by extra-MBH brain regions. In summary, the present study indicates that the CS secretory response to 2-DG is mediated by adenohypophyseal ACTH and that 2-DG induces hyperadrenocortical function and hyperglycemia by acting upon CNS sites located outside the MBH region.
113 REFERENCES 1 Berthoud, H. R. and Baetting, K., Effect of insulin and 2-deoxy-D-glucose on plasma glucose level an lateral hypothalamic eating threshold in the rat, Physiol. Behav., 12 (1973) 547-556. 2 Chevrier, C., Hammiche, N. and Brudieux, R., Effects of 2-deoxy-D-glucose on plasma and adrenal concentrations of corticosterone and aldosterone in standard diet fed and potassium loaded rats, Horm. Metabol. Res., 14 (1982) 94-97. 3 Dallman, M. F. and Yates, F. E., Dynamic asymmetries in the corticosteroid feedback pattern and distribution-metabolism-binding elements of adrenocortical system, Ann. N.Y. Acad. Sci., 156 (1969) 696-721. 4 Feldman, S., Conforti, N., Chowers, I. and Davidson, J. M., Differential effects of hypothalamic deafferentation on responses to different stresses, lsr. J. reed. Sci., 4 (1968) 908-911. 5 Halasz, B. and Gorski, R. A., Gonadotrophic hormone secretion in female rats after partial or total interruption of neural afferents to the medial basal hypothalamus, Endocrinology, 80 (1967) 608-622. 6 Muller, E. E., Cocchi, D. and Forni, A., A central site for the hyperglycemic action of 2-deoxy-D-glucose in mouse
and rat, Life Sci., 10 (1971) 1057-1067. 7 Roth, J., Glick, S. M., Yalow, R. S. and Berson, S. A., The influence of blood glucose on plasma concentration of growth hormone, Diabetes, 13 (1962) 355-361. 8 Siegel, R., Chowers, I., Conforti, N. and Feldman, S., Corticotrophin and corticosterone secretory patterns following acute neurogenic stress, in intact and in variously hypothalamic deafferentated male rats, Brain Research, 188 (1980) 399-410. 9 Smith, G. P. and Epstein, A. N., Increased feeding in response to decreased glucose utilization in the rat and monkey, Amer. J. Physiol., 217 (1969) 1083-1087. 10 Sun, C. L., Thoa, N. B. and Kopin, I. J., Comparison of the effects of 2-deoxyglucose and immobilization on plasma levels of catecholamines and corticosterone in awake rats, Endocrinology, 105 (1979) 306-310. 11 Weidenfeld, J., Siegel, R. A., Feldman, S., Conforti, N. and Chowers, I., ACTH and corticosterone secretion following insulin in intact and in variously hypothalamic deafferentated male rats, Exp. Brain Res., 48 (1982) 306-308. 12 Yasuda, N., Takebe, K. and Greer, M. A., Evidence of nycterohemeral period in stress-induced pituitary-adrenal activation, Neuroendocrinology, 21 (1976) 214-224.