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Am J Physiol Endocrinol Metab 290: E243-E250, 2006. First published September 6, 2005; doi:10.1152/ajpendo.00118.2005
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Hypothalamic paraventricular nucleus, but not vasopressin, participates in chronic hyperactivity of the HPA axis in diabetic rats

Dóra Zelena,1 Ludmila Filaretova,1,3 Zsuzsa Mergl,1 István Barna,1 Zsuzsanna E. Tóth,2 and Gábor B. Makara1

1Institute of Experimental Medicine, Hungarian Academy of Sciences; 2Laboratory of Neuromorphology, Semmelweis University Medical School, Budapest, Hungary; and 3Laboratory of Experimental Endocrinology, Pavlov Institute of Physiology, St. Petersburg, Russia

Submitted 17 March 2005 ; accepted in final form 1 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Diabetes mellitus (DM), as chronic stress activates the hypothalamo-pituitary-adrenocortical axis. We examined whether arginine vasopressin (AVP) and the hypothalamic paraventricular nucleus (PVN) participate in DM-induced chronic stress symptoms. AVP-deficient Brattleboro or PVN-lesioned Wistar rats were used with heterozygous or sham-operated controls. The rats were studied 2 wk after a single injection of streptozotocin. The appearance of DM (enhanced water consumption and blood glucose elevation) and the chronic stress-like somatic changes (body weight decrease, thymus involution, adrenal gland hypertrophy) were not influenced by the lack of AVP. By contrast, PVN lesion significantly attenuated DM-induced thymus involution and adrenal gland hypertrophy as well as the increase in water consumption. The corticotropin-releasing hormone mRNA in PVN was diminished by DM and elevated by the lack of AVP without interaction. DM elevated the proopiomelanocortin (POMC) mRNA in the anterior lobe of the pituitary. The lack of AVP had no effect, whereas lesioning the PVN significantly diminished the elevation. The elevated basal corticosterone plasma levels detectable in DM were influenced neither by the lack of AVP nor by lesioning the PVN. Thus the lack of AVP had no influence on DM-induced chronic stress symptoms, but lesioning the PVN attenuated part of them. However, the lack of elevation in POMC mRNA after PVN lesion, together with the maintained corticosterone elevation, suggests that direct adrenal gland activation occurs in untreated DM.

chronic stress; Brattleboro rats; corticosterone; proopiomelanocortin; corticotropin-releasing hormone; diabetes mellitus; hypothalamo-pituitary-adrenocortical axis

DIABETES MELLITUS (DM) is an endocrine disorder that is quite common in developed countries (17). Inadequate insulin secretion and insulin resistance with inadequate compensatory insulin release are the main reasons for DM development. The use of toxic agents that destroy {beta}-cells in the pancreas and lead to insulin deficiency, such as streptozotocin (STZ), produces a model of DM in animals (36). The most frequent symptoms of DM are blood glucose level elevation, polyuria, polydipsia, and weight loss. However, the long-term disorder can cause many other complications, including cardiovascular, cerebrovascular, or renal ones.

Long-term DM as a chronic stress may also induce a dysfunction of the hypothalamo-pituitary-adrenocortical (HPA) axis. A tight connection between HPA axis activity and blood glucose regulation is well known (9, 12). The HPA axis is responsible for the production of glucocorticoids, which are important adaptive hormones. Glucocorticoid hormones participate in maintaining the normal blood glucose level that is especially important for the brain. Hypoglycemia is a strong stimulus for HPA axis activation and glucocorticoid production (11, 19). Hyperactivity of the HPA axis in experimental STZ-induced DM had already been found in 1976 (16). It is also known that patients with both types of DM can have an increased plasma cortisol level (12). Acute increases in plasma glucocorticoid levels are useful during stress conditions (20, 21, 38). However, chronic increase in plasma glucocorticoid concentration can be harmful in general (37) and also for diabetic patients because these hormones might aggravate the disorder by further elevating of blood glucose levels and inducing insulin resistance (2). In general, chronic glucocorticoid elevation can cause diseases, but chronic diseases keep the body in constant stress, too. DM creates stressful conditions for organisms as well as adrenal hyperactivity and diminished feedback control, which are associated with a stressful environment, too, and are further risk factors for DM (7) and may result in a vicious circle. It has also been shown (41) that, in chronic STZ-diabetes rats, the central responsiveness of the HPA system to stimulatory and/or inhibitory inputs is also changed in conjunction with changes in glucocorticoid feedback sensitivity.

Acute and chronic stressful stimuli activate the HPA system. Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are the main neuropeptide hormones of the hypothalamic paraventricular nucleus (PVN) involved in activating this system (3, 40). These peptides stimulate the secretion of adrenocorticotropin (ACTH), as well as the synthesis and processing of proopiomelanocortin (POMC) to ACTH in corticotroph cells of the anterior lobe (AL) of the pituitary. ACTH reaches the adrenal cortex and stimulates the synthesis and release of glucocorticoids (in rats, this is predominantly corticosterone). Inactivation of the stress response is achieved via glucocorticoid negative feedback. It has been proposed that, during chronic stressful stimulation there is a shift in favor of a preferential activation of AVP rather than CRH release from the hypothalamus. The relative resistance to feedback inhibition of ACTH release stimulated by AVP might explain how chronic stressful stimulation maintains high adrenocortical hormone levels (1, 13, 31, 40).

The mechanisms providing hyperactivation of the HPA axis in diabetes is still unclear. The role of AVP in the process might be suspected, as the osmotic diuresis resulting from hyperglycemia is bound to enhance AVP secretion (8). Moreover, AVP is supposed to have a preferential role during chronic stress situations, even after repeated insulin treatment (15). The PVN is the main hypothalamic center of the HPA axis during acute stress stimuli (33); however, its role during chronic stress situations is not so clear. Previous studies (39) suggested that, during DM, HPA activation with a central component in the PVN occurs.

Thus the aim of this study was to investigate the role of AVP and/or PVN in DM-induced HPA dysfunction. In case AVP and/or the PVN are instrumental for the maintenance of adrenocortical responsiveness during DM-induced chronic stress, then the AVP-deficient Brattleboro rat (47) and/or PVN-lesioned animals (33) are expected to show markedly impaired changes during STZ-induced DM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male rats were used throughout the experiments. Brattleboro rats were maintained at our institute in a colony started from breeder rats from Harlan (Indianapolis, IN). We compared the AVP-deficient homozygous (di/di) rats with diabetes insipidus to heterozygous (di/+) control rats from the same litters (46). Wistar rats were obtained from Charles River Hungary (Budapest, Hungary). Rats were kept in a controlled environment (23 ± 1°C, 50–70% humidity, 12 h of light starting at 0700) and given commercial rat chow (Charles River Hungary) and tap water ad libitum. The animals were kept one per cage from the beginning of the experiment. Decapitation was carried out between 9:00 and 12:00 in the morning. The experiments were performed in accordance with regulations set by the Hungarian Council for Animal Care and were supervised by the Institutional Animal Care and Use Committee.

Experiments

In experiment 1, male Brattleboro rats were injected intravenously with STZ (60 mg/kg; Sigma-Aldrich, Budapest, Hungary) or citrate buffer vehicle (pH 5) through the tail vein under short restraint (maximum 2 min) and left undisturbed (except weekly body weight measurement and daily bedding change) for 14 days. The mortality was 5.5% for heterozygous (di/+) and 5.3% for homozygous (di/di) STZ-treated animals. Rats were killed by decapitation on the 15th day. The experiments were conducted in three series. The average initial weights were di/+: 312.6 ± 13 g (n = 34) and di/di: 288.2 ± 7 g (n = 44).

In experiment 2, male Wistar rats were anesthetized with intraperitoneal ketamine-xylazine cocktail, and the PVN was lesioned with a wire knife, using a stereotaxic frame as described earlier (33, 45). Control rats were subjected to sham operation. Five days later, STZ or vehicle was injected through the tail vein. Fourteen days after STZ injection (on day 20 after PVN lesion) the animals were decapitated. From PVN-lesioned animals, only rats showing a distinct and complete lesion of the PVN on histological examination were retained for later analysis. The mortality after STZ injection was 22.7% for sham-operated and 25.5% for PVN-lesioned rats because of the combined effect of surgery (anesthesia and skull opening, not the lesion) and DM. The PVN lesion studies were performed in four series. The average initial weight of the animals was 286.7 ± 3 g (n = 81).

Blood Glucose

Appearance of diabetes was monitored by daily measurement of water consumption and confirmed by measuring the blood glucose concentration from trunk blood by use of an analyzer developed for animal laboratories. Useful range was from 1.1 to 30 mmol/l, and the sensitivity of the method was 0.1 mmol/l.

Hormone Analysis

Trunk blood was collected at the end of 2 wk on K2-EDTA-containing tubes on ice. After centrifugation, the plasma was stored at –20°C until hormone measurement. Plasma ACTH was measured by radioimmunoassay (RIA) in 50 µl of unextracted plasma, as described earlier (44). The intra- and interassay coefficients of variation were 6.4 and 17.7%, respectively. Plasma corticosterone was measured from 10 µl of unextracted plasma by RIA using a specific antiserum developed at our institute (45). The intra- and interassay coefficients of variation were 6.39 and 16.6%, respectively. The neurointermediate lobe (NIL) of the pituitary was homogenized in 100 µl of 0.1 N HCl, and after centrifugation the supernatant was kept at –20°C until AVP and oxytocin (OT) contents were measured by specific RIAs. Anti-AVP and -OT antisera were produced in rabbits and obtained from Dr. M. Vecsernyés (Szent-Györgyi Medical University, Szeged, Hungary). 125I-labeled tracers were produced by the chloramine-T method. Bound and free fractions were separated by charcoal in the AVP RIA and by a second antibody in the OT RIA. The intra- and interassay coefficients of variation for the AVP-RIA were 5.42 and 19%, and for the OT RIA they were 5.12 and 14.28%, respectively.

In Situ Hybridization

Tissue preparation. The brains were removed and frozen at –80°C in n-hexane and stored until cryostat sectioning. Frozen forebrains were mounted on a cryostat-microtome and cut in 16-µm sections. Every sixth 16-µm section was mounted on a silanized slide from the anterior comissure to the end of the amygdala. Pituitaries were quickly dissected from decapitated animals, and the separated ALs were frozen at –80°C in embedding medium. These specimens were mounted on a cryostat-microtome and cut in 12-µm sections.

Hybridization process. The hybridization technique was derived from that described by Simmons (42). Tissue sections were fixed with 4% paraformaldehyde and then digested with proteinase K (1 µg/ml in 50 mM Tris, pH 8, and 5 mM EDTA), acetylated (0.25% acetic anhydride in 0.1 M triethanolamine at pH 8), and dehydrated using ascending ethanol series (50–100%). The hybridization mixture (50% formamide, 0.3 M NaCl, 10 mM Tris, pH 8, 2 mM EDTA, 1x Denhardt’s, 10% dextran sulfate, 0.5 mg yeast tRNA, 107 dpm/ml specific probe) was pipetted onto the slides and left overnight at 58°C. On the next day, the slides were rinsed four times with SSC (1x SSC: 0.15 M, 15 mM trisodium citrate, pH 7) digested with ribonuclease A (10 µg/ml in 5 mM Tris, 1 mM EDTA, 0.5 M NaCl, pH 8), gradually desalted, and washed in 0.1x SSC at 65°C for 30 min. After a final wash in 0.1x SSC at room temperature for 1 min, slides were air dried for imaging plate exposition or further dehydrated for emulsion autoradiography.

CRH mRNA. CRH mRNA levels were quantified by [35S]UTP containing riboprobes complementary to the exonic sequences of the CRH gene (the plasmid containing the 1.2-kb template was a generous gift of Dr. K. Majo, Northwestern University).

After the hybridization process, slides were exposed to imaging plates (Fujifilm, BAS-IP, MS 2340) for 72 h, and the plates were scanned by a fluorescent image analyzer (FLA 3000, Fujifilm, scanning resolution of 50 µm). Radiograms were evaluated by using the public domain National Institutes of Health (NIH) Image program (written by Wayne Rasband at NIH and available from the Internet by anonymous ftp from zippy.nimh.nih.gov). The boundary of the examined region was outlined, and the average grayness value was corrected by the background taken from the neighboring hypothalamic tissue. mRNA expression was evaluated by summing up the grayness values measured over the whole extent of the PVN (integrated density, 6.44).

In experiment 1, integrated density for amygdala centralis (CeA) was also calculated. In addition, the extension of the signal was also reviewed. We counted how many sections of one brain showed the CHR mRNA signal. From these data, we calculated the anteroposterior extension; e.g., if we found 10 sections with a distinguishable CRH mRNA signal, that meant 10 (number of slides) x 6 (every 6th section was collected) x 16 (thickness of one slide) = 960 µm, the length of CRH mRNA contaning brain area.

In experiment 2, in addition to the aforementioned analysis, the hybridized slides were dipped into Kodak NTB3 nuclear emulsion and exposed for 3 days. The intensity of the signal above the CeA was measured through a microscope by using a threshold tool of the NIH Image program. The threshold tool segmented the image into features of interest and background. Pixels with brightness values greater than or equal to the lower threshold and less than or equal to the upper threshold were displayed in red. The red area was divided by the number of cells and are displayed in Table 1 as a signal/cell. Cells were definied as having grains three standard deviations above background. In case of doubt, the slide was examined under normal lighting conditions with Giemsa stain. These measurements were done on two sections of the left and right CeA. Values of four CeA cross sections were averaged (34). The number of cells was not different between groups (the range was from 12.1 to 13.7 cells/slide).


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  		Table 1. CRH mRNA levels in CeA

 
POMC mRNA. POMC mRNA levels were quantified by riboprobes complementary to the exonic sequences of the POMC gene labeled by [35S]UTP (the plasmid containing the 1.2-kb template was a generous gift of Dr. J. Eberwine, University of Pennsylvania). After the hybridization process, the slides were exposed to imaging plates, and the evaluation was performed as described above for CRH mRNA hybridization.

Statistical Analysis

Values are presented as means ± SE. Data were analyzed by two-way analysis of variance (ANOVA) using the ANOVA/multiple ANOVA module of the Statistica 6.0 software package (Tulsa, OK). Water consumption was analyzed by repeated-measures ANOVA. Multiple pairwise comparisons were made by the Newman-Keuls method.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Role of Vasopressin in DM-Induced Chronic Stress: Studies of Brattleboro Rats in Experiment 1

Appearance of DM. The di/di animals had inborn diabetes insipidus, which was already shown by the enhanced water consumption before STZ injection (di/+: 35 ± 2 ml; di/di: 180 ± 18 ml, n = 39–36; Fig. 1). DM-induced increased water consumption was also present in our experiments. It had already started on the day after STZ injection and stabilized 6 days later. STZ injection was able to induce further water intake in di/di animals, parallel with the changes in di/+ animals. Blood glucose levels at 15 days were elevated by previous STZ injection, and these changes were the same in di/+ and di/di animals.



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  		Fig. 1. Appearance of diabetes mellitus (DM) in Brattleboro rats. A: water consumption, n = 17–21 rats. All effects and interaction (except genotype x DM) were significant. B: blood glucose, n = 8–15. Effect of DM was significant (P < 0.01). C, nondiabetic group, control; DM, streptozotocin-induced DM; di/+, heterozygous control rats; di/di, arginine vasopressin-deficient homozygous Brattleboro rats. *P < 0.05 vs. day 1 (A); **P < 0.01 vs. nondiabetic (B); #P < 0.05 vs. nondiabetic; {dagger}P < 0.05 vs. sham operated.

 
Somatic changes. Initial body weight was smaller in di/di than in di/+ rats (di/+: 312.6 ± 13 g; di/di: 288 ± 7 g, P < 0.01; Fig. 2). Both the STZ-induced DM and the lack of AVP resulted in slower body weight gain, and these effects were additive. The percentage of body weight changes were: di/+ nondiabetic group, control (C): 13.2 ± 1.9; di/+ DM: –7.6 ± 1.9; di/di C: 8.09 ± 1.7; di/di DM: –15 ± 2.5. We show relative organ weights instead of absolute weights because the initial body weight differences could mask or intensify some changes. Reduced thymus weights occurred in DM rats. The lack of AVP had no effect on its own and did not modify the STZ-induced thymus weight decrease. Similar, but opposite changes were present in adrenal weights. STZ-induced DM increased the weight of the adrenal glands as a typical sign of chronic stress. This elevation was not influenced by the lack of AVP in di/di animals.



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  		Fig. 2. DM-induced somatic changes in Brattleboro rats, n = 17–25. A: body weight changes. Effects of genotype and DM were significant (P < 0.01) without interaction. B: relative thymus weight. C: relative adrenal gland weight. Effect of DM was significant in B and C (P < 0.01). **P < 0.01 vs. nondiabetic rats; ##P < 0.01 vs. di/+ rats.

 
HPA axis and amygdala changes. CRH mRNA in the PVN was higher in nonstressed di/di than in di/+ animals (Fig. 3). Two weeks of DM decreased the CRH mRNA levels not only in di/+ but also in di/di rats. The lack of AVP did not affect the DM-induced changes. POMC mRNA levels in the AL were not influenced by the lack of AVP, but DM induced similar elevations in both genotypes (effect of DM, P < 0.001). Plasma ACTH levels were influenced neither by genotype nor by DM (Table 2). Plasma corticosterone levels were almost the same in di/+ and di/di animals, and DM caused an elevation in both genotypes (effect of DM, P = 0.003; effect of genotype, P = 0.22, no interaction). In the CeA, both the genotype and the DM significantly enhanced the intensity or extension of the CRH mRNA signal, although in some instances just a borderline difference was present (Table 1). These effects seem to be additive.



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  		Fig. 3. Hypothalamo-pituitary-adrenocortical (HPA) axis changes during DM in Brattleboro rats. A: CRH mRNA levels in paraventicular nucleus (PVN), n = 9–17. Effects of genotype (P = 0.022) and DM (P < 0.01) were significant without interaction. B: proopiomelanocortin (POMC) mRNA in the anterior lobe (AL), n = 5–10. C: plasma corticosterone levels, n = 8–11. Effect of DM was significant in B and C (P < 0.01). *P < 0.05; **P < 0.01 vs. nondiabetic rats.

 

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  		Table 2. Plasma ACTH levels

 
Changes in the NIL. AVP and OT content of the NIL was decreased by DM (Table 3). In di/di animals the AVP was undetectable, whereas the OT content was decreased and DM induced a further decrease.


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  		Table 3. Neurohormone content in the neurointermediate lobe of the pituitary

 
Role of PVN in DM-Induced Chronic Stress: Studies with PVN Lesion in Experiment 2

Appearance of DM. In sham-operated Wistar rats, the STZ injection induced a fast increase in water consumption (Fig. 4). In PVN-lesioned rats, the STZ-induced increase in water consumption began later, and the rise did not reach the level of sham-operated animals. Elevated blood glucose levels were measured on day 15. Lesioning the PVN did not influence the STZ-induced glucose elevation, which was approximately threefold.



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  		Fig. 4. Appearance of DM in PVN-lesioned rats. A: water consumption, n = 11–19. All effects and interaction were significant (P < 0.01). B: blood glucose, n = 8–13. Effect of DM was significant (P < 0.01). Sh, sham operated; PVNL, PVN lesion. *P < 0.05; **P < 0.01 vs. day 1 (A) or nondiabetic animals (B); #P < 0.05, ##P < 0.01 vs. nondiabetic rats; +P < 0.05, ++P < 0.01 vs. sham-operated rats.

 
Somatic changes. Body weight gain was reduced by DM to the same extent in sham-operated and PVN-lesioned rats (Fig. 5). The weight of thymus was reduced in STZ-induced DM. PVN lesion alone increased thymus weights, but the decreasing effect of DM was still present, albeit to a lesser extent than in sham-operated animals. Adrenal weights were elevated in DM rats and PVN-lesion decreased the weights, but DM induced further, albeit smaller, elevation in PVN-lesioned rats.



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  		Fig. 5. DM-induced somatic changes in PVN-lesioned rats, n = 17–25. A: body weight change. Initial body weight was 290 ± 3.6 g. Effect of DM was significant (P < 0.01). B: relative thymus weight. C: relative adrenal weight. All effects and interaction were significant in B and C (P < 0.01). **P < 0.01 vs. nondiabetic animals; #P < 0.01 vs. sham-operated animals.

 
HPA axis and amygdala changes. As a tendency, the CRH mRNA in PVN was smaller in DM animals, although this tendency did not reach significance in the Wistar rats (P = 0.068; Fig. 6). POMC mRNA signal in the AL was elevated in DM animals. The PVN lesion had no effect on basal POMC mRNA levels but diminished the DM-induced POMC elevation. Plasma ACTH levels were influenced by neither the lesioning of the PVN nor the STZ-injection (Table 2). Plasma corticosterone levels were at least doubled in DM animals. The PVN lesion had no effect on basal corticosterone levels and failed to influence the corticosterone-elevating effect of DM. Integrated CRH mRNA signal in amygdala had a tendency for higher intensity after PVN lesion with no effect of DM. On emulsion-dipped slides, the effect of PVN lesion on the CeA CRH mRNA was significant (P = 0.049; Table 1).



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  		Fig. 6. HPA axis changes during DM in PVN-lesioned rats. A: CRH mRNA levels in PVN. Effect of DM was almost significant (n = 5–6, P = 0.068). B: POMC mRNA in the AL, n = 11–17. Effect of DM (P < 0.01) and DM x PVNL interaction (P = 0.04) were significant. C: plasma corticosterone levels, n = 8–13. Effect of DM was significant (P < 0.01). **P < 0.01 vs. nondiabetic animals; ##P < 0.01 vs. sham-operated animals.

 
Changes in NIL. In DM animals, the levels of AVP and OT in the NIL were decreased (Table 3). After PVN lesion, the levels of both hormones were decreased and DM induced further decreases in PVN-lesioned animals, but the combined PVN lesion-DM effect was smaller than could be expected on additivity (DM x PVN lesion interaction, P = 0.051).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study was designed to test the participation of AVP and PVN (the origin of CRH and AVP) in DM-induced chronic stress symptoms. The results show that, in DM-induced chronic stress the lack of AVP had no influence on the symptoms, although we should take into consideration the limitation of the knockout model (47). PVN mediates DM-induced thymus involution, adrenal gland hypertrophy, and POMC mRNA increase in the AL. At the same time, the contrasting finding that PVN lesion induced inhibition of POMC mRNA but maintained plasma corticosterone elevation suggests that, during DM, direct adrenal gland activation also probably occurs through peripheral mediators.

We (43) used a single injection of 60 mg/kg STZ and studied stress parameters at 15 days before adaptation could have occured, at around day 30. High water consumption and blood glucose levels confirmed that DM was induced. An interesting finding was that the high water consumption in diabetes insipidus (di/di) animals was further increased by DM, so the diuresis due to AVP deficiency was additive, with the osmotic diuresis caused by DM. This fact can have special importance in some clinical situations, for example, during gestation or in diabetic nephropathy (5, 18), because the consequences of a combination of DM and diabetes insipidus can be extremely severe (14). Therefore, diabetes insipidus (incidence rate: ~1 in 6,666 in the US) seems to be an additional serious risk factor for DM and thus a potential target for prevention and/or therapeutic intervention. It was also surprising that lesioning the PVN diminished the water consumption in DM because of the partial lack of AVP [approximately one-half of the circulating AVP comes from the PVN, whereas the other half comes from the supraoptic nucleus (SON)]; opposite changes could be expected. It is possible that a reduction during the first week in daily production of the diabetogenic hormone corticosterone can reduce the appearance of DM in PVN-lesioned animals. However, after 2 wk, the blood glucose elevation and the morning plasma corticosterone levels were the same in sham-operated and PVN-lesioned rats.

Body weight reduction (23, 27), thymus involution (41), and adrenal gland hypertrophy (23, 39) are typical signs of chronic stress. Glucocorticoid overexposure probably suppresses the immune system and reduces thymus weights in DM rats. None of the chronic stress-induced somatic changes were influenced by the AVP deficiency in the Brattleboro rats. DM was also able to influence the chronic stress signs after PVN lesion, but at an attenuated level. Although the basal morning corticosterone levels 2 wk after STZ seems not to have been influenced by PVN lesion, the smaller thymus enlargement and adrenal hypertrophy suggest that the overall corticosterone supply during this 2 wk of DM was reduced in PVN-lesioned rats. The smaller body weight gain in control Brattleboro rats, compared with Wistar rats from experiment 2, suggests different metabolic regulation of the two strains. It should also be noted that, in the di/di Brattleboro rats, besides the behavioral ingestive defects, the water balance deficit can lead to energy substrate loss in the urine, which is demonstrated by larger body weight loss. Therefore, the metabolic cost of DM seems to be different in the two models.

The CRH mRNA-decreasing effect of DM was present in both the Wistar and Brattleboro rats and appears to be a consequence of the maintained high corticosterone levels during untreated DM. In case the glucocorticoid hormone stimulation during DM is a consequence of a yet unknown mediator acting at the adrenal cortical level and not through the PVN, a hypothalamic CRH mRNA decrease might then be a secondary consequence of the negative feedback action of glucocorticoids on the CRH/AVP-producing neurons. In addition, the presence of a CRH mRNA decrease in DM animals with di/di genotype suggests that this decrease might not be due to the AVP elevation often seen in chronic stress situations. The CRH mRNA elevation in the PVN of di/di animals is a novel finding that could be a direct consequence of the lifelong absence of AVP in the HPA axis regulation. Because AVP and CRH potentiate each other’s actions on ACTH secretion, in the absence of one of them the other might compensate, as was already shown in the CRH knockout mice, where AVP mRNA was elevated (34).

Chronic stresses, including DM, may induce various changes in the endocrinological, behavioral, and emotional balance of the organism. The amygdala (as part of the limbic system) plays an important role in emotions and is in tight connection with HPA axis regulation. Previous studies (4, 25, 30) strongly support the role of the amygdalar CRH system in behavioral and/or emotional responses to stress. The slightly and/or significantly enhanced intensity and extended area of the CRH mRNA signal in the CeA of the AVP-deficient and DM animals suggest that both the mutation-induced diabetes insipidus and DM might lead to a predisposition to anxiety or emotional imbalance (Table 1). The findings in Wistar rats with bilateral PVN lesions (Table 1) are consistent with the observations of Palkovits et al. (35) that lesioning the PVN eliminates the inhibitory tone on the CeA CRH system.

ACTH secretion may change fast, and its instantaneous level in plasma does not necessarily reflect chronic changes in the HPA axis and is normal in experimental DM rats (23). The chronic influences on the HPA system are better shown by the ACTH precursor POMC mRNA level in the AL, which changes slowly and appears to integrate influences over several hours; therefore, its level better reflects long-term changes. POMC mRNA in the AL showed elevation after STZ-induced DM. This is consistent with other chronic stress models (1), although it is in contrast with a report on POMC mRNA reduction 2 wk after 75 mg/kg intraperitoneal STZ (28) or an absence of change 8 days after 65 mg/kg STZ (10). The discrepancy could be due to important methodological differences. Kim et al. (28) measured POMC mRNA in the whole pituitary, where the signal from the NIL is ~10 times higher than that from the AL, and the measurement probably reflects the opioid POMC peptide mRNA changes in the intermediate lobe. Chan et al. (10) used cannulated animals, and the slight chronic stress involved in chronic cannulation procedure might increase the control levels of POMC mRNA and thus might have obscured an increase due to DM.

The interesting contrast of CRH mRNA reduction in the PVN, along with an elevation in POMC mRNA in the AL, suggests interplay of other factors. Although CRH is supposed to be the main ACTH releasing hormone, there are several other possible factors [oxytocin (29), serotonin, angiotensin, vasoactive intestinal peptide, cholecystokinin, neuropeptide Y (8a)] that can induce POMC mRNA and ACTH elevations, and, as Schwartz et al. (39) already proposed, this mechanism could become more important in HPA axis regulation during uncontrolled DM. We suggest that the PVN has to be the origin of the possible HPA axis activation during DM because of the lesioning in the PVN diminishing the POMC elevation. A major candidate for HPA axis regulation would be AVP acting in synergism with CRH, but we can exclude it as an indispensable factor because in di/di rats the lack of AVP did not reduce the POMC elevation. Higher CRH receptor level in AL during chronic stress could also take part in maintaining ACTH responses in the absence of AVP (26).

Basal corticosterone plasma levels were also elevated in DM rats (27, 39). Not only the average corticosterone level but also the circadian corticosterone rhythmicity can be disturbed in the 15-day DM rat (23, 43). The lack of AVP had no effect on CRH mRNA in PVN, POMC mRNA in AL, or morning corticosterone elevation in DM-induced chronic stress, which suggests that AVP does not play a crucial role in HPA axis regulation during DM-chronic stress. By contrast, lesioning in the PVN diminished the AL POMC mRNA, but not plasma corticosterone elevation in DM animals. Taken together, these findings suggest two separate pathways for adrenal gland activation during DM, one through PVN and another through a peripheral mechanism that can activate the adrenal gland directly, bypassing hypothalamic PVN and the AL. During DM, many humoral factors can be released into the general circulation (12), the sensitivity of the adrenal gland can be changed (41), and/or the role of direct adrenal gland innervation (22) can come to the fore.

In our experiments, the AVP and OT contents of the NIL of the pituitary were decreased in DM animals. Water loss in di/di and/or DM animals can stimulate AVP secretion, deplete the AVP and OT stores of the NIL of the pituitary, and at the same time stimulate their synthesis, which may be reflected in an increase in the neuronal cell bodies in the PVN and SON (32). The PVN is one of the two nuclei that contain a large amount of AVP and OT projecting to NIL, so it is no surprise that after PVN lesion, the AVP and OT content of NIL were decreased.

The apparent contradictions in the various reports might be reconciled by suggesting that the uncontrolled diabetes may induce dynamic changes in the HPA system in which hypothalamic, pituitary, and adrenal changes all participate at various degrees in a time-dependent fashion. It appears that a hypothalamic or suprahypothalamic stimulation is present in the first 2 wk after induction of experimental STZ-DM, which results in increases in POMC mRNA and consequent ACTH synthesis. ACTH stimulates the adrenal cortex, increases plasma glucocortioid levels, and may contribute to hyperglycemia. In addition to that sequence, our findings suggest that an unidentified (metabolic?) component in DM might stimulate the adrenal gland directly and increase secretion of glucocorticoids. Excess glucocorticoids in the plasma might provide negative feedback to the hypothalamus and decrease activation of the whole HPA system.

In conclusion, our data suggest that AVP does not play a crucial role in HPA axis regulation during DM-chronic stress. By contrast, our findings support previous suggestions that the PVN participates in the realization of some DM-induced chronic stress symptoms. Apart from PVN-mediated adrenal gland activation in DM, there is also another, PVN-independent way that provides stimulation of glucocorticoid production, probably through peripheral mediators.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by Hungarian Grant ETT 044-99 to D. Zelena, OTKA T-025845, T-032915, and ETT 276-2000 to G. B. Makara, and Russian Foundation Basic Research (RFBR) Grant No. 04-04-48507, Science School Grant No. 1163-2003-4, and a grant for Centre of Excellence from the European Community to the Institute of Experimental Medicine, No. ICA1-CT-2000-70004.


   ACKNOWLEDGMENTS
 
We thank Hajni Beko for excellent assistance with the animals, and the Hormone Laboratory of the Institute of Experimental Medicine for the hormone analyses. F. A. László (University of Szeged, Szeged, Hungary) provided valuable help by establishing the Brattleboro breeding colony.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Zelena, Institute of Experimental Medicine, H-1083 Budapest, Szigony 43, Hungary (e-mail: zelena{at}koki.hu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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A. Domokos, Z. Mergl, I. Barna, G. B Makara, and D. Zelena
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