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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Departments of Physiology (O.C., K.I., M.V., S.G.M.), Obstetrics and Gynecology (S.G.M.), and Medicine (M.V.), University of Toronto, Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Dr. Stephen G. Matthews, 1 King’s College Circle, Medical Sciences Building Room 3240, University of Toronto, Toronto, Ontario, Canada, M5S 1A8. E-mail: . stephen.matthews{at}utoronto.ca
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Abstract |
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Introduction |
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Increased HPA function in type 1 and type 2 diabetic patients is characterized by elevated circulating cortisol levels and increased 24-h urinary free cortisol levels (2). Moreover, diabetic patients exhibit disrupted circadian patterns of cortisol secretion, with elevated cortisol levels during the nadir and normal or slightly elevated values during peak secretion (1, 3). It has been suggested that such augmentation in HPA activity is due to altered control of ACTH release from pituitary corticotrophs and/or the direct stimulatory actions of CRH at the adrenal gland, independent of pituitary ACTH release (4, 8). Many type 1 and type 2 diabetic patients exhibit nonsuppression of pituitary-adrenal activity following glucocorticoid administration, compared with nondiabetic individuals (9), which suggests that glucocorticoid negative feedback is also impaired in these patients.
The purpose of this study was to 1) characterize circadian variations in the diabetic HPA axis; 2) determine mechanisms involved in responses to stress; and 3) establish whether changes in basal activity and stress responsiveness are the result of alterations in pituitary, adrenal and/or glucocorticoid negative feedback sensitivity. As we previously observed differences in HPA function during the morning hours, we evaluated stress responsiveness, pituitary and adrenal sensitivity and glucocorticoid negative feedback at 1000 h.
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Materials and Methods |
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Three groups of rats were used: 1) normal controls (n = 6), 2) untreated STZ-induced diabetic (n = 6) and 3) ip insulin-treated diabetic rats (n = 6). On d 0, indwelling SILASTIC brand tubing (length 3 cm; inner diameter, 0.020 in.; outer diameter, 0.037 in.; Dow Corning Corp., Midland, MI) connected to a polyethylene catheter (length 10 cm; PE-50, Clay Adams, Boston, MA) was inserted into the left carotid artery for blood sampling and right jugular vein for injection, under ketamine (100 mg/kg body weight, ip; MTC Pharmaceuticals, Cambridge, Ontario, Canada); acepromazine (1 mg/kg body weight, ip; Wyeth-Ayerst Laboratories Canada Inc., Montréal, Québec, Canada); xylazine (1 mg/kg body weight, ip; Bayer Corp. Inc., Etobicoke, Ontario, Canada) cocktail anesthesia using aseptic techniques. A heparin-saline solution (10 U/ml) was used to prime the catheters to prevent blood clot formation. The catheters were tunneled sc, exteriorized through a skin incision, and anchored to the back of the neck between the scapulae, through a steel tether (Lomir, Québec, Canada). The tether was then connected to a rodent swivel system (Lomir) located outside of the cage to minimize investigator interaction. The catheters were aspirated and reprimed daily with 1% heparinized-saline to maintain patency.
Diabetes was induced at the end of the surgery with the administration of a single ip injection of STZ (65 mg/kg; Sigma, St. Louis, MO). Control animals received a sterile saline injection under similar conditions. Animals treated with STZ were given 10% sucrose in their drinking water for the first 24 h following the STZ injection to prevent hypoglycemia (7). This model of type 1 diabetes mellitus features moderate diabetes with fasting hyperglycemia and normal plasma insulin during fasting but reduced plasma insulin during feeding (10). A subgroup of diabetic rats received Linplants (
2.5 U of insulin per day, ip; LinShin Canada, Inc., Ontario, Canada), a sustained release bovine insulin preparation, 4 d after the induction of diabetes under light ketamine/acepromazine/xylazine anesthesia. This form of insulin treatment results in plasma insulin concentrations of approximately 650 pM, which is comparable to fed-state insulin levels (7). Technical aspects of the insulin implant have been detailed previously (11). Blood glucose was monitored twice daily with blood glucose meters (Glucometer Elite 3903; Bayer Corp. Inc.) in all animals to ensure that normoglycemia was maintained in the control and insulin-treated diabetic groups, and that adequate hyperglycemia (>15 mM) was achieved in the uncontrolled diabetic group in the postprandial period. In addition, the insulin-treated diabetic rats were frequently monitored to ensure that hypoglycemia did not occur. Animals that experienced hypoglycemic episodes were excluded from the study.
Basal activity
Eight days following catheterization, arterial blood samples were taken to examine plasma ACTH and corticosterone concentrations at 0800 h, 1300 h, and 1800 h. At each sampling point, the contents of the arterial catheter were aspirated, 0.6 ml of blood was then withdrawn and the catheter was flushed with 1% heparinized saline.
For the measurement of ACTH, blood was collected from the carotid artery catheter into chilled tubes containing EDTA (Sangon Limited Canada, Scarborough, Ontario, Canada) and Trasylol (Bayer Corp. Inc.). Serum was collected for corticosterone measurements. Plasma and serum samples were separated, aliquoted into storage tubes, and stored at -20 C until assayed. The packed red blood cells from ethylenediamine-tetraacetic acid ACTH samples were resuspended in a warm, sterile, cell-free artificial plasma solution containing 4% BSA (12) and reinfused after each blood sampling to prevent volume depletion and anemia.
Restraint stress
On d 10, the animals were subjected to 20 min of restraint stress in a clear acrylic tube (Harvard Apparatus, Holliston, MA). Basal blood samples were taken at 1000 h (0 min), before the rats being restrained, and subsequently at 5, 15, 30, 60, and 120 min. Red blood cells were reinfused into the rat after each sampling as described above.
Low-dose CRH, ACTH, and dexamethasone suppression tests
Twelve, 14, and 16 d after surgery, the animals were administered a low-dose of rat CRH (0.5 µg/kg; Peninsula Laboratories, Inc.; San Carlos, CA), human ACTH1–24 (75 ng/kg; Peninsula Laboratories, Inc.) or dexamethasone (25 µg/kg; Sigma) through the jugular vein catheter, respectively, to evaluate pituitary, adrenal and glucocorticoid negative feedback sensitivity. A 48-h recovery period was allowed between each test. Basal blood samples for the assessment of plasma ACTH and serum corticosterone concentrations were taken at 1000 h (0 min), immediately before the administration of CRH, ACTH1–24, or dexamethasone. In all cases, a submaximal dose was used to assess target tissue sensitivity, as opposed to glandular hormone storage capacity (4). Blood samples were subsequently taken at 15, 30, 60, 120, and 180 min after administration. In preliminary studies, we noted that this iv injection protocol did not significantly stress the animals (plasma ACTH responses to injection of saline at 0, 15, 30, 60, 120, and 180 min: 60.2 ± 3.6, 71.5 ± 8.5, 69.2 ± 5.8, 68.7 ± 8.8, 87.9 ± 10.4 and 76.4 ± 4.6 pg/ml; plasma corticosterone responses to injection of saline at 0, 15, 30, 60, 120, and 180 min: 51.1 ± 2.4, 55.8 ± 2.0, 54.0 ± 9.5, 56.5 ± 4.9, 60.0 ± 3.6, and 54.5 ± 5.2 ng/ml). The hematocrit, determined at the beginning and at the end of each experiment, was maintained above 35% throughout the course of the study.
Plasma hormone determination
Plasma ACTH (DiaSorin, Inc., Stillwater, MN) and corticosterone (ICN Pharmaceuticals, Inc., Orangeburg, NY) concentrations were determined using commercially available RIA kits as described previously (7).
Data analysis
Hormone data are presented as mean ± SEM. For measurements taken repeatedly in all groups, a two-factor (group x time) ANOVA was conducted. For measurements taken on one occasion only (i.e. body weight, basal hormones etc.), a one-factor (group) ANOVA was performed. If a significant F-ratio was found, comparisons between mean values were made using Tukey’s HSD post hoc test. These analyses were performed using the Statistical Analysis System package for personal computers (SAS Institute, Inc., Cary, NC) with P < 0.05 set as the criterion for statistical significance.
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Results |
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). Blood glucose was significantly (P < 0.05) elevated in diabetic animals compared with both normal and insulin-treated diabetic animals (Table 1). Although initial hematocrit levels were significantly (P < 0.05) elevated in diabetic and insulin-treated diabetic rats compared with normal rats (normal: 41.6 ± 1.3, diabetic: 47.0 ± 1.6, and insulin-treated diabetic: 49.5 ± 0.8%), there was no significant drop in hematocrit levels in any of the treatment groups during each of the studies.
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). Insulin treatment restored both ACTH and corticosterone concentrations to normal. No significant differences in plasma ACTH or corticosterone levels were observed between the three treatment groups at either 1300 h or 1800 h.
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and Tables 2 and 3). Insulin treatment of diabetic rats partially restored ACTH (P < 0.05) and fully restored corticosterone responses to restraint stress.
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and Tables 2
and 3). Following the iv administration of rat CRH (0.5 µg/kg), control and insulin-treated diabetic groups exhibited significant activation of pituitary-adrenal activity (Fig. 3 and Tables 2 and 3). The initial rise in plasma ACTH following CRH administration was significant (P < 0.05) in normal and insulin-treated animals, but not in uncontrolled diabetic animals. Net plasma corticosterone responses were significantly (P < 0.05) greater in control and insulin-treated diabetic groups but again, not in uncontrolled diabetic animals.
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and Table 3). After the iv administration of human ACTH1–24 (75 ng/kg), we noted that the rise in plasma corticosterone was significantly (P < 0.05) impaired in uncontrolled diabetic animals compared with normal and insulin-treated diabetic animals (Fig. 4 and Table 3). Corticosterone concentrations in normal and insulin-treated diabetic animals rose significantly (P < 0.01) in the first 30 min following the administration of ACTH. No significant change was seen in the uncontrolled diabetic animals.
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and Tables 2 and 3). This was followed by a gradual recovery of both ACTH and corticosterone levels. Plasma corticosterone concentrations at 180 min were significantly (P < 0.05) elevated in control and insulin-treated diabetic animals compared with uncontrolled diabetic rats. Pituitary-adrenal activity of uncontrolled STZ-diabetic animals, however, was not significantly suppressed following dexamethasone administration.
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Discussion |
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Basal pituitary-adrenal activity appears to be elevated during the trough of the circadian rhythm in untreated diabetic rats and these plasma ACTH and corticosterone concentrations are similar to previously reported values (16). The high plasma corticosterone levels are maintained throughout the course of the day whereas in normal and insulin-treated diabetic animals, the levels tend to increase later in the day. Throughout the study, plasma ACTH and corticosterone concentrations tend to increase around 1200 h and this is likely due to the fact that although rats consume the majority of their food during the dark cycle, they were consistently observed to be feeding at this time. It has previously been reported that plasma ACTH and corticosterone concentrations tend to rise just before feeding bouts (17, 18).
Despite significantly elevated basal plasma ACTH and corticosterone concentrations, the pituitary-adrenal response of diabetic rats to restraint stress was greatly diminished in comparison to control and insulin-treated diabetic animals. This is in contrast to the augmented response to histamine injection in STZ-diabetic rats reported by Scribner et al. (13). Inconsistencies between our results and those of Scribner are likely attributable to the application of different stressors that act via different central pathways to activate the HPA axis to varying degrees. However, our results are quantitatively similar to the impaired HPA response to insulin-induced hypoglycemia that we have reported previously in STZ-diabetic rats (19). The lower plasma corticosterone response is consistent with lower plasma ACTH concentrations. Insulin treatment of diabetic animals, however, only partially restored the ACTH response to immobilization. Despite this, the corticosterone response was normalized in this treatment group, suggesting that insulin may play a role in increasing adrenocortical responsiveness to ACTH (20).
The issue of whether pituitary sensitivity is affected by diabetes is still controversial. In humans, varied ACTH responses to CRH challenge have been reported in patients with diabetes. These include unaltered (2), reduced (4) and increased (21) ACTH secretion following CRH administration to diabetic patients. In these studies, a 1 µg/kg dose of CRH was used to ascertain corticotroph sensitivity. Coiro et al. (4) indicated that this higher dose of CRH is more suitable for the assessment of pituitary ACTH storage capacity, whereas a lower dose similar to that used in the present study is more appropriate for assessing pituitary sensitivity. With a low-dose CRH stimulation test, Coiro demonstrated that the rise in ACTH was significantly lower in diabetic patients. This is consistent with the impaired adrenocorticotrophic response seen in STZ-diabetic rats in the present study. In uncontrolled STZ-diabetic animals, pituitary responsiveness to CRH administration was significantly lower compared with the control group, despite the animals having elevated pituitary-adrenal activity under basal conditions. This provides support for the hypothesis that basal hyperactivation of the diabetic HPA axis may be the result of increased hypothalamic drive and not altered pituitary sensitivity. In addition, this observation is consistent with the impaired response to restraint demonstrated by these animals. In a previous study, using the STZ-diabetic rat model, impaired responsiveness to a CRH challenge was correlated with a decreased number of CRH receptors in the anterior pituitary, and it was suggested that this resulted from the hypersecretion of hypothalamic CRH (13). However, it is not known whether such decreases in CRH receptors occur in diabetic patients. In our hands, insulin treatment restored pituitary ACTH responsiveness to CRH stimulation. Plasma corticosterone concentrations rose significantly in the normal and insulin-treated diabetic groups following CRH administration, but not in the untreated diabetic group. The reduced plasma corticosterone response to CRH suggests that pituitary corticotroph sensitivity may be altered in STZ-diabetes. In contrast to our findings, Scribner et al. (13) reported no changes in pituitary sensitivity of pentobarbital-anesthetized STZ-diabetic rats. This may be attributed to the use of anesthesia, along with a high dose of CRH in the latter study.
To assess adrenocortical function, we used a low-dose ACTH stimulation test. Following administration of 75 ng/kg of ACTH, uncontrolled diabetic animals did not exhibit a significant rise in corticosterone levels when compared with control and insulin-treated diabetic groups. This indicates that adrenal sensitivity is not increased in uncontrolled STZ-diabetic animals. Scribner et al. (13) demonstrated that adrenal responsiveness to ACTH did not change in sodium pentobarbital-anesthetized STZ-diabetic rats. This may be due to differences in the dose of ACTH used and/or the fact their studies were performed in the afternoon—a time when adrenal sensitivity has been shown to be increased (22). Furthermore, a previous study performed in diabetic patients reported that the administration of ACTH revealed low adrenocortical responsiveness (23). Taken together, the evidence in both human and animal studies, along with the data presented in our current and previous studies (7), indicate that hyperactivation of the HPA axis in diabetes is partially mediated by increased hypothalamic drive, whereas decreased stress responsiveness in diabetes is likely the result of decreased pituitary and adrenocortical sensitivity.
To evaluate glucocorticoid negative feedback sensitivity, we used the low-dose dexamethasone suppression test. In a pilot study, we tested varying doses of dexamethasone ranging from 10–500 µg/kg and determined that 25 µg/kg was the optimal dosage to evaluate both suppression and recovery of HPA activity within the 3-h duration of the experiment. Following iv administration of dexamethasone, we noted significant suppression of pituitary-adrenal activity in normal and insulin-treated diabetic rats, but not in uncontrolled diabetic animals. These results indicate that glucocorticoid negative feedback sensitivity is decreased in the early stages of STZ-diabetes, and that sensitivity is restored with insulin treatment. Nonsuppression of pituitary-adrenal activity following a dexamethasone suppression test has been reported in both type 1 and type 2 diabetic patients (9). Studies in type 1 diabetic patients have also demonstrated that this deterioration in feedback sensitivity is exacerbated with increased duration of diabetes (24) and that it may (9) or may not (25) be related to glycemic control. Furthermore, we previously demonstrated that hippocampal, hypothalamic, and pituitary glucocorticoid receptor mRNA levels are unaltered in STZ-diabetic rats, whereas hippocampal mineralocorticoid receptor mRNA levels are significantly increased (7). An increase in corticosteroid receptors normally results in increased inhibitory tone on the axis (26, 27, 28). However, dexamethasone nonsuppression and the fact that hypothalamic CRH mRNA, plasma ACTH, and corticosterone concentrations are all elevated in STZ-diabetes, suggests that there may be an increase in hypothalamic drive and/or impaired glucocorticoid negative feedback may be involved in HPA hyperdrive in STZ-diabetes. Further studies will have to be performed to determine the nature of the impaired feedback—whether it is due to a decrease in glucocorticoid receptor binding affinity or due to a defect in the glucocorticoid receptor-signaling pathway. Insulin treatment restored glucocorticoid negative feedback sensitivity. This latter observation may be due to the increase in anterior pituitary GR mRNA expression that we have reported previously (7) or normalization of plasma corticosterone concentrations. Increased GR expression may result in greater suppression of ACTH synthesis and/or secretion.
In conclusion, our results indicate that hyperactivation of the HPA axis in early STZ-diabetes is likely caused by both an increase in central drive (7) and a decrease in glucocorticoid negative feedback sensitivity, whereas impaired responsiveness to stress in STZ-diabetic rats likely involves a decrease in sensitivity of the pituitary corticotroph and adrenal cortex to CRH and ACTH, respectively. More importantly, normalization of pituitary-adrenal activity in STZ-diabetic rats with insulin therapy can be attributed, in part, to restoration of insulin concentrations or pituitary-adrenal function. These impairments in HPA function in diabetes may contribute to cognitive dysfunction (29, 30), decreased counterregulation to hypoglycemia (31), and an impaired ability to respond to stress.
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Acknowledgments |
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Footnotes |
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Abbreviations: HPA, Hypothalamo-pituitary-adrenocortical; STZ, streptozotocin.
Received August 16, 2001.
Accepted for publication January 25, 2002.
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, P < 0.05 vs. normal. 
, P < 0.01 vs. normal.
