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Endocrinology, doi:10.1210/en.2007-1473
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Endocrinology Vol. 149, No. 6 2990-3001
Copyright © 2008 by The Endocrine Society

Adaptation to Mild, Intermittent Stress Delays Development of Hyperglycemia in the Zucker Diabetic Fatty Rat Independent of Food Intake: Role of Habituation of the Hypothalamic-Pituitary-Adrenal Axis

Holly E. Bates, Adam S. Sirek, Michael A. Kiràly, Jessica T. Y. Yue, Danitza Goche Montes, Stephen G. Matthews1 and Mladen Vranic1

Departments of Physiology (H.E.B., A.S.S., M.A.K., J.T.Y.Y., D.G.M., S.G.M., M.V.), Obstetrics and Gynecology (S.G.M.), and Medicine (S.G.M., M.V.), Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence to: Holly Bates, Room 3363 Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: holdoug{at}yahoo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypothalamic-pituitary-adrenal (HPA) axis hyperactivity occurs in type 2 diabetes, and stress is assumed to play a causal role. However, intermittent restraint stress, a model mimicking some mild stressors, delays development of hyperglycemia in Zucker diabetic fatty (ZDF) rats. We examine whether such stress delays hyperglycemia independent of stress-induced reductions in hyperphagia and is due to adaptations in gene expression of HPA-related peptides and receptors that ameliorate corticosteronemia and thus hyperglycemia. ZDF rats were intermittently restraint stressed (1 h/d, 5 d/wk) for 13 wk and compared with obese control, pair fed, and lean ZDF rats. After 13 wk, basal hormones were repeatedly measured over 24 h, and HPA-related gene expression was assessed by in situ hybridization. Although restraint initially induced hyperglycemia, this response habituated over time, and intermittent restraint delayed hyperglycemia. This delay was partly related to 5–15% decreased hyperphagia, which was not accompanied by decreased arcuate nucleus NPY or increased POMC mRNA expression, although expression was altered by obesity. Obese rats demonstrated basal hypercorticosteronemia and greater corticosterone responses to food/water removal. Basal hypercorticosteronemia was further exacerbated after 13 wk of pair feeding during the nadir. Importantly, intermittent restraint further delayed hyperglycemia independent of food intake, because glycemia was 30–40% lower than after 13 wk of pair feeding. This may be mediated by increased hippocampal MR mRNA, reduced anterior pituitary POMC mRNA levels, and lower adrenal sensitivity to ACTH, thus preventing basal and stress-induced hypercorticosteronemia. In contrast, 24-h catecholamines were unaltered. Thus, rather than playing a causal role, intermittent stress delayed deteriorations in glycemia and ameliorated HPA hyperactivity in the ZDF rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WIDELY ASSUMED that stress worsens type 2 diabetes mellitus (T2DM). This assumption does not consider the variability, complexity, or divergence between effects of chronic and intermittent stress and results from the paucity of adequately controlled human studies examining these in the context of T2DM.

Stress acutely increases alertness, suppresses appetite and growth, and raises glycemia by promoting gluconeogenesis, insulin resistance, and reduced insulin secretion (1). These effects are mediated by hypothalamic-pituitary-adrenal (HPA) and sympatho-adrenomedullary system activation (1). Stress induces CRH secretion from the hypothalamic paraventricular nucleus (PVN), stimulating proopiomelanocortin (POMC) synthesis and processing in the anterior pituitary into ACTH (2, 3). During chronic stress, arginine vasopressin (AVP) secretion from the PVN is also an important ACTH secretagogue (4, 5). ACTH stimulates synthesis and secretion of corticosterone in rodents (cortisol in humans) (6). The HPA response is terminated by glucocorticoid negative feedback through occupation of type 1 [mineralocorticoid receptor (MR)] and type 2 [glucocorticoid (GR)] corticosteroid receptors (7).

Chronic stress causes adaptations within the HPA axis that protect the organism from chronic hypercorticosteronemia that include habituation of the corticosterone (8, 9) and catecholamine responses (10) and reduced or absent CRH responses to homotypic stressors (11, 12), although AVP continues to be able to respond (5, 9). There are often adaptive increases in basal nadir corticosterone levels (13) and basal AVP (11, 14), which may be related to altered glucocorticoid feedback inhibition of the HPA axis (15, 16). Thus, the response to intermittent stress is more complex than under acute conditions.

We recently demonstrated that intermittent restraint stress, an activator of the HPA and sympatho-adrenomedullary axes (17), surprisingly delays development of hyperglycemia in the Zucker diabetic fatty (ZDF) rat while normalizing basal corticosterone measured at study termination (18). Similar glycemic benefits were observed in ZDF rats in response to intermittent water exposure (19) and in Otsuka Long Evans Tokushima fatty (OLETF) rats with repeated immobilization (20) and suggest that intermittent exposure to mild stressors may be beneficial for delay of hyperglycemia. However, the mechanism for these effects is unclear, and in previous studies, food intake in pair-fed rats did not adequately mimic that of stressed animals (18) or pair-fed control rats were not included (20), and therefore, it has not been possible to distinguish between the effects of stress-induced reductions in food intake on glycemia and the effects of intermittent stress per se (18).

In the current study, we hypothesized that 1) adaptation to intermittent stress would delay hyperglycemia partially independent of reductions in hyperphagia, 2) normalization of basal corticosterone after 13 wk of intermittent stress would be consistent throughout a 24-h day and thus not result from alterations in corticosterone circadian rhythms, 3) reductions in basal corticosterone after 13 wk of intermittent stress will be associated with down-regulation in gene expression within the HPA axis, and 4) down-regulation of basal corticosterone and HPA axis gene expression will occur independent of reductions in hyperphagia.

These hypotheses were examined in the ZDF rat model of T2DM, which has a pathogenesis that closely resembles that of T2DM in humans. This genetic model develops hyperglycemia when consuming normal rat chow (18), and therefore, effects of intermittent stress on glycemia and the HPA axis that occur independent of stress-induced reductions in food intake could be examined, unlike other models of diet-induced obesity/insulin resistance. The ZDF rat expresses a truncated, nonfunctional leptin receptor, making them resistant to the effects of leptin (21, 22, 23), which causes their obesity, insulin resistance, and hyperinsulinemia. This leptin resistance is similar to that in obese humans (24), albeit to a greater severity. Finally, use of a rat model allows control of the type, predictability, and severity of the stressor, variables that have confounded human stress studies in the past. Pair-fed obese ZDF control rats had their food intake matched to that of intermittently restraint-stressed obese ZDF rats treated for 13 wk to delineate the effects of intermittent stress on glycemia and HPA parameters that occur independent of food intake. To our knowledge, this is the first study to examine the relationship between the HPA axis and intermittent stress per se with development of hyperglycemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care and maintenance
Male obese ZDF (ZDF/Crl-Leprfa/fa) or lean (ZDF/Crl-LeprFa/?) rats obtained at 5 wk old from Charles Rivers (Wilmington, MA) were individually housed in opaque microisolation cages in temperature- (22–23 C) and humidity-controlled rooms. Rats were kept on a 12-h light, 12- h dark cycle (0700–1900 h) and fed normal chow (Harlan 2018) in wire lids. All experiments were performed according to protocols approved by the University of Toronto Animal Care Committee and followed guidelines from the Canadian Council for Animal Care.

Study design (Fig. 1Go)
Lean (Fa/?) ZDF rats (Ln-control, n = 9) and obese (fa/fa) ZDF rats (n = 13–14 per group) were obtained in seven batches at 5 wk old and acclimatized to the light cycle, food, and handling for 1 wk. From 6 wk old, obese (Ob) ZDF rats were randomly divided into three treatment groups: 1) intermittent stress (Ob-stress), 2) intermittent food/water restriction (Ob-FWR control), and 3) intermittent food/water restriction and pair fed (Ob-FWR pair fed). Intermittently stressed rats were restraint stressed for 1 h/d, 5 d/wk between 0900 and 1200 h in Broome rodent restrainers (Harvard Apparatus, Saint Laurent, Quebec, Canada) for 13 wk. To mirror the loss of food and water by intermittently stressed rats during this time, Ob-FWR control, Ob-FWR pair fed, and Ln-FWR control rats had their food and water removed for 1 h/d, 5 d/wk during the same time period and thus underwent intermittent food/water restriction. To control for the reduction in food intake that occurs with intermittent stress, each Ob-FWR pair fed rat was staggered 1 wk behind a paired Ob-stress rat and had their available food matched to that eaten by an Ob-stress rat during the same week of treatment. Food intake was matched on a weekly basis because we previously demonstrated that daily pair feeding induced hoarding behavior and resulted in inadequate pair feeding (18).


Figure 1
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  		FIG. 1. Study design. A, ZDF (ZDF/Crl-Leprfa) rats (n = 13–14 per group) and ZDF lean (+/?) rats (n = 9) were obtained at 5 wk old and acclimatized by daily handling for 1 wk. At 6 wk old, obese ZDF rats were restraint stressed for 1 h/d, 5 d/wk for 13 wk (Ob-Stress) and compared with age-matched ZDF obese diabetic control rats (Ob-FWR Control) and age-matched ZDF nondiabetic, nonobese lean rats (Ln-FWR Control) that had food and water removed for 1 h/d, 5 d/wk. ZDF obese pair-fed rats were included to control for the effect of stress on food intake (Ob-FWR Pair Fed) and had their food intake restricted on a weekly basis to match that of stressed rats and had food and water removed for 1 h/d, 5 d/wk for 13 wk. B, After 12 wk of treatment, rats were chronically cannulated. After 2 d recovery, treatment resumed for 2 d. On the third day of the 13th week of treatment, basal samples were taken every 2 h for corticosterone and every 6 h for ACTH and catecholamines. The following week, animals were euthanized for organ retrieval 24 h after their last treatment.

 
For 12 wk, body weight and food intake were monitored weekly. Morning fed glycemia was monitored twice weekly between 0900 and 1100 h before treatment and 16–18 h fasting glycemia was monitored once weekly also between 0900 and 1100 h before treatment. Glycemic responses to treatment (restraint stress in Ob-stress rats or food/water removal in Ln-FWR control, Ob-FWR control, and Ob-FWR pair-fed rats) were measured once weekly concurrent with measurements of basal and posttreatment corticosterone levels obtained between 0900 and 1200 h. Blood for glucose (<2 µl) and corticosterone (~20 µl) was taken by tail nick as described (18).

We previously demonstrated normalization of a single measurement of basal nadir corticosterone after 13 wk of intermittent restraint (18). Therefore, the current study examined diurnal variation in basal hormones over a 24-h period after 12 wk of treatment by surgical insertion of chronic indwelling catheters into the left carotid artery and right jugular vein. Catheters were inserted into the carotid artery for blood sampling and into the jugular vein for reinfusion of red blood cells after larger blood samples every 6 h. Treatment was resumed after 3 d recovery (Fig. 1BGo). Five days after surgery, basal samples were obtained every 2 h starting at 0700 h for glucose, insulin, and corticosterone (150 µl blood) in EDTA/K-coated microvettes (Sarstedt, Montreal, Quebec, Canada) through cannulas or tail nick when cannulas were nonfunctional. Every 6 h starting at 0900 h, ACTH and catecholamine samples were obtained (1 ml blood) in 1:1 EDTA (Sigma-Aldrich, Oakville, Ontario, Canada)/Trasylol (Bayer Canada Ltd., Mississauga, Ontario, Canada). Red blood cells were resuspended in 1% heparinized saline (100 U/ml) and reinfused into the rat to prevent blood loss. Plasma samples were stored at –80 C until analysis.

Animals were euthanized 24 h after treatment during the 14th week of treatment. Adrenal glands, thymus, and epididymal fat pads were weighed. The brain and pituitary gland were quickly removed under sterile conditions, frozen on dry ice, and stored at –80 C until processing for in situ hybridization.

Assays
Plasma insulin was assayed using a rat insulin ELISA kit (Crystal Chem, Downers Grove, IL), and corticosterone (MP Biomedicals, Orangeburg, NY), ACTH (MP Biomedicals), and catecholamines (Labor Diagnostika Nord GmbH & Co., Nordhorn, Germany) were assayed using RIA kits described previously (18, 19, 25).

Surgeries
Vessel cannulations were performed using aseptic techniques under isoflurane anesthesia (Aerrane; Baxter, Mississauga, Ontario, Canada) after 12 wk of treatment, as described (26). Buprenorphine (0.01 mg/kg sc) (Schering-Plough, Pointe-Claire, Quebec, Canada) was given to aid postsurgical recovery. Catheters were exteriorized through a rodent-swivel system (Lomir, Quebec, Canada), minimizing investigator interaction. Patency of the jugular catheter was maintained by slow infusion of 0.1% heparinized saline (10 U/ml, 0.37 µl/kg·h) at a nondetrimental rate (27). Carotid catheters were flushed daily with 1% heparinized saline (100 U/ml). Food was supplied as a combination of rodent meal and pellets after surgery to facilitate feeding in the presence of the swivel system.

In situ hybridization
Methods for in situ hybridization were described (28). Briefly, pituitary and coronal brain cryosections (10 µm) from the PVN, arcuate nucleus, and hippocampus were obtained according to stereotaxic coordinates (29). Twelve pituitary and six brain sections were analyzed per rat for each probe. Approximately 45-mer antisense oligonucleotide probes [CRH (bases 536–580), AVP (bases 588–632), POMC (bases 572–616), MR (bases 2942–2986), and GR (bases 1321–1365) (Dalton Chemical Laboratories, Toronto, Ontario, Canada) and NPY (bases 184–228) (Sigma-Genosys, The Woodlands, TX)] were labeled with [35S]deoxyadenosine 5'({alpha}-thio)-triphosphate (dATP, 1250 Ci/mmol; PerkinElmer, Waltham, MA) and applied to each slide. Slides were incubated overnight at 42.5 C, washed in standard saline citrate, and dehydrated in 70 and 95% ethanol. Slides were exposed to autoradiographic film (Biomax; Eastman Kodak, Rochester, NY) (CRH, 5 wk; AVP, 1d; arcuate nucleus POMC, 2 wk; pituitary anterior lobe POMC, 1d; pituitary intermediate lobe POMC, 2 h; arcuate nucleus NPY, 2 wk; MR, 2 wk; and GR, 4 wk) and the relative OD (ROD) of signal quantified after subtraction of background values using an image analysis system (Imaging Research, St. Catherines, Ontario, Canada). The ROD of each probe for each rat was calculated based on the average of the six brain or 12 pituitary sections.

Statistics
Data are presented as means ± SEM. Statistical analyses were performed with Statistica 6.0 (Statsoft, Tulsa, OK), and P < 0.05 was considered significant. Repeated-measures ANOVA or factorial ANOVA were used when appropriate and the Newman-Keuls post hoc test for multiple comparisons performed if the ANOVA revealed a P < 0.05. One-factor analyses were done by unpaired Student’s t test for comparison of obese to lean ZDF rats or by one-way ANOVA between the three obese ZDF groups to examine treatment effects. Area under the curve (AUC) for 24-h hormones was calculated using the trapezoidal rule from 0 using Origin 6.0 (Microcal Software Inc., Northampton, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Feeding
Food intake (Fig. 2AGo) and body weight (Fig. 2BGo).
Obese groups ate more than lean rats (treatment P < 0.0001; post hoc P < 0.0005). In obese rats, intermittent stress reduced hyperphagia 5–20% (treatment P < 0.0001; post hoc P < 0.0005), and therefore, pair-fed rats had a matched reduction in hyperphagia (treatment P < 0.0001; post hoc P < 0.0005). Obese groups had higher body mass than lean rats (treatment P < 0.0001; post hoc P < 0.0005), although neither intermittent stress nor pair feeding lowered body mass or epididymal fat mass in obese rats (Table 1Go). Thus, we examined weekly body weight gain when normalized to food intake (supplemental Fig. 1Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). This body weight gain changed over time (treatment x time P = 0.003) such that control rats gained less weight per gram of food eaten than did pair-fed and stressed rats during the 10th and 12th weeks of treatment.


Figure 2
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  		FIG. 2. Food intake (A), body weight (B), and in situ hybridization of arcuate nucleus neuropeptide mRNA expression 24 h after the last treatment (C). Values are expressed as mean ± SEM. Statistics to the right of the curve represent main effects of treatment with Newman Keuls post hoc test; n = 9–14 for food intake and body weight; n = 7–9 for arcuate nucleus neuropeptide mRNA. *, P < 0.05 vs. lean; {dagger}, P < 0.05 vs. control. L, Ln-FWR control; C, Ob-FWR control; PF, Ob-FWR pair fed; S, Ob-stress.

 

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  		TABLE 1. Body and organ weights after 13 wk of intermittent restraint stress

 
Arcuate nucleus POMC and NPY mRNA expression 24 h after treatment (Fig. 2CGo).
As expected in obese groups, POMC mRNA expression, the precursor for anorexigenic {alpha}-MSH, was reduced 40% (P < 0.006 vs. lean), and orexigenic NPY mRNA was increased 50% (P < 0.05 vs. lean). In obese groups, neither POMC nor NPY mRNA expression were altered by intermittent stress, but POMC mRNA was reduced by pair feeding (P < 0.05 vs. control).

Glycemia
Glucose response to treatment (Fig. 3AGo).
Glucose responses to 1 h restraint stress (Ob-stress) or food/water removal (Ln-FWR control, Ob-FWR control, Ob-FWR pair-fed groups) were measured. Glucose responses were affected by treatment over time (P < 0.0001). Restraint stress increased glycemia in the first week compared with all obese and lean rats from whom food and water was removed (P < 0.05). Responses habituated thereafter to comparable levels induced by food/water removal in lean rats. Interestingly, glycemic responses to food/water removal in control and pair-fed rats increased over time relative to lean rats from 6 or 9 wk onward, respectively (P < 0.05), suggesting that obese rats are hyperresponsive to stress.


Figure 3
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  		FIG. 3. Glucose response to restraint stress in Ob-stress rats or food and water removal in Ln-FWR control, Ob-FWR control, and Ob-FWR pair-fed rats (A), development of morning fed hyperglycemia (B), and development of fasting hyperglycemia (C). A, Restraint stress initially elicits a glucose response, which habituates over time. Treatment was restraint stress in stressed rats or food and water removal in control groups. B, Intermittent stress attenuates the rise in morning fed glucose compared with control rats and compared with pair feeding. Values are expressed as mean ± SEM. Statistics to the right of the curve represent main effects of treatment with Newman Keuls post hoc test; n = 9–14 per group. *, P < 0.05 vs. Ln-FWR control; {dagger}, P < 0.05 vs. Ob-FWR control; §, P ≤ 0.05 vs. Ob-FWR pair fed.

 
Development of hyperglycemia (Fig. 3Go, B and C).
Morning glycemia was elevated in obese control compared with lean rats (treatment P < 0.0001; post hoc P < 0.05) from the third week onward (P < 0.05). In obese rats, intermittent stress attenuated the rise in morning glycemia compared with obese food/water-restricted control rats (treatment P < 0.0001; post hoc P < 0.0005) to 30% lower levels by 13 wk (P < 0.05). Intermittent stress also lowered morning glycemia during the 12th and 13th weeks of treatment compared with pair feeding in obese rats (Figs. 3BGo and 4AGo), indicating food intake-independent effects of intermittent stress.


Figure 4
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  		FIG. 4. Basal glycemia, insulinemia, and relative insulinemia over 24 h (A–C) and during the light and dark phase (D–F) during the 13th week of treatment. Thirteen weeks of intermittent restraint stress (P < 0.0001) but not pair feeding ameliorates 24-h hyperglycemia. This is accompanied by greater relative hyperinsulinemia during the light phase but is not independent of the reduction in food intake. Data are presented as mean ± SEM; n = 5–11 per group or time point. Brackets indicate differences between lights on and lights off by paired Students t test. *, P ≤ 0.05 vs. Ln-FWR control; {dagger}, P ≤ 0.05 vs. Ob-FWR control; §, P < 0.05 vs. Ob-FWR diet restricted.

 
Fasting glycemia was elevated in obese control relative to lean rats during all weeks (treatment P < 0.0001; post hoc P < 0.0005). In obese rats, intermittent stress attenuated fasting hyperglycemia compared with control rats (treatment P < 0.001; post hoc P < 0.01) to 50% lower levels after 12 wk (P < 0.005). Pair feeding also ameliorated fasting hyperglycemia (treatment P < 0.0001; post hoc P < 0.03 vs. control). However, by the 13th week, fasting glycemia was lowered by intermittent stress compared with both ZDF obese control and pair-fed rats (P < 0.005), whereas fasting glycemia was no longer reduced by pair feeding (lean, 5.2 ± 0.2; control, 14.2 ± 1.8; pair-fed, 13.6 ± 2.0; and stress, 7.7 ± 1.2 mmol/liter), indicating food intake-independent effects of intermittent stress.

Glycemia (24-h) and insulinemia after 13 wk of treatment (Fig. 4Go).
Basal glycemia and insulinemia after 13 wk of treatment were higher in all obese ZDF groups compared with lean rats over 24 h (treatment P < 0.0001; post hoc P < 0.005). In obese rats, glycemia was reduced by intermittent stress compared with both obese food/water-restricted control and pair-fed rats (treatment P < 0.0001; post hoc P < 0.005). This was associated with higher relative insulinemia, as shown by an approximately 2.5-fold increase in the ratio of insulin to glucose during the light phase (P = 0.05). In contrast, pair feeding did not significantly lower 24-h glycemia or increase insulinemia. However, relative insulinemia after 13 wk of intermittent stress was similar to that induced by pair feeding. Thus, the amelioration of hyperglycemia with intermittent stress that is independent of reductions in food intake is not caused by higher insulinemia.

Adaptations in the HPA axis
Adrenal and thymus weights (Table 1Go).
Body mass at euthanasia and epididymal fat mass were not affected by intermittent stress or pair feeding in obese rats, suggesting proportional changes in body mass in obese groups. Neither intermittent stress nor pair feeding caused adrenal hypertrophy or atrophy of the thymus.

Basal corticosterone (Fig. 5Go, A and B).
Basal corticosterone increased over time (time P < 0.0001) but was higher in obese than lean rats (treatment P < 0.0001; post hoc P < 0.0005) during all weeks (P < 0.05). In obese rats, basal corticosterone was not affected by intermittent stress or pair feeding over time, although by 12 wk of treatment, hypercorticosteronemia was exacerbated in pair-fed compared with intermittently stressed and control rats (P < 0.03).


Figure 5
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  		FIG. 5. Weekly and 24-h corticosterone levels. Basal hypercorticosteronemia in obese ZDF rats is exacerbated by the 12th week of pair feeding (A) and during the corticosterone nadir after 13 wk of pair feeding (B). Corticosterone responses (C) and absolute corticosterone levels (D) after restraint (Ob-Stress) habituate over time in comparison with responses to food and water removal (Ob-FWR control and pair-fed groups) over 12 wk of treatment (treatment x time P < 0.01). Data are presented as mean ± SEM. Statistics to the right of the curve represent main effects of treatment with Newman Keuls post hoc analysis. *, P < 0.05 vs. lean; {dagger}, P < 0.05 vs. control; §, P ≤ 0.05 vs. pair fed; #, P < 0.05 nadir vs. trough, paired t test. L, Ln-FWR control; C, Ob-FWR control; P, Ob-FWR pair fed; S, Ob-stress.

 
During wk 13, basal corticosterone was sampled from cannulas every 2 h over 24 h. Compared with lean rats, pair feeding of obese rats increased basal corticosterone levels (P < 0.001, 24-h corticosterone AUC). This differed from the basal hypercorticosteronemia observed in all obese rats during weekly samples and likely occurred because many 24-h samples from lean rats were repeatedly obtained by tail nick (four of nine rats), likely elevating corticosterone levels. Indeed, an unpaired Student’s t test comparing the mean corticosterone levels obtained by tail nick vs. a catheter revealed that levels obtained by tail nick were significantly higher (tail, 550.1 ± 55.6 nmol/liter; catheter, 332.2 ± 36.3 nmol/liter; P = 0.001). Similar to weekly basal samples, the 24-h basal corticosterone AUC was elevated by pair feeding (P < 0.01 vs. control) but not intermittent stress (P = 0.02 vs. pair fed).

We arbitrarily chose the corticosterone nadir to be 0300–0900 h, and the peak 1500–2100 h, based on the corticosterone curves (supplemental Fig. 1Go). In obese rats, after 13 wk of pair feeding, basal hypercorticosteronemia was apparent during the nadir (P < 0.01 vs. control) but not the peak. Intermittent restraint prevented this nadir hypercorticosteronemia (P = 0.02 vs. pair fed). Pair feeding additionally resulted in a loss of normal diurnal corticosterone rhythms (P = 0.24, paired t test, nadir vs. peak), which was not observed in obese control or stressed rats (P < 0.05). Diurnal corticosterone variation was not apparent in lean rats, likely because of the high proportion of samples obtained by tail nick.

Stress-induced corticosterone (Fig. 5Go, C and D).
Corticosterone responses to restraint stress (Ob-stress) or food/water removal (Ln-FWR control, Ob-FWR control, Ob-FWR pair fed) were examined weekly (Fig. 5CGo). Lean rats showed a slight response to food/water removal and the sampling procedure that habituated over time. This response to food/water removal was potentiated in obese rats (treatment P = 0.02, Ln-FWR control vs.Ob-FWR control), illustrating that obese ZDF rats are hyperresponsive to stress. When the response to restraint stress in intermittently stressed obese rats was compared with that of intermittent food/water removal, the corticosterone response markedly habituated over time as expected with intermittent exposure to a homotypic stressor compared with both control (treatment x time P = 0.007. Ob-stress vs. Ob-FWR control) and pair-fed rats (treatment x time P < 0.005, Ob-stress vs.Ob-FWR pair fed) (Fig. 5CGo). Thus, restraint induced higher corticosterone levels than food/water removal in Ob-FWR control and/or Ob-FWR pair-fed rats for the first 3 wk (Fig. 5DGo), which declined thereafter. These habituated levels between 8 and 12 wk of treatment not only were lower than levels induced by food/water removal in obese, intermittently food- and water-restricted rats (Ob-FWR control and Ob-FWR pair fed) but also resulted in comparable levels as those induced by food/water removal in lean ZDF rats.

Basal ACTH (Fig. 6AGo) and pituitary POMC mRNA (Fig. 6BGo).
Basal ACTH levels were measured every 6 h over 24 h after 13 wk via cannulas (supplemental Fig. 2). ACTH was not altered in obese compared with lean rats. Similarly, in obese rats, ACTH was not altered by intermittent stress or pair feeding. However, when we examined the ratio of corticosterone to ACTH, an index of adrenal sensitivity to ACTH, this ratio was elevated in obese rats more than 5-fold by pair feeding and 3-fold by intermittent stress during the corticosterone nadir (0900 h) (P < 0.01 vs. obese-FWR control). However, the corticosterone to ACTH ratio was further elevated by pair feeding in comparison with intermittent restraint (P = 0.05).


Figure 6
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  		FIG. 6. The 24-h ACTH (A) and basal anterior pituitary POMC mRNA expression measured by in situ hybridization (B) after 13 wk of intermittent restraint stress. A, The ratio of corticosterone to ACTH during the corticosterone nadir (0900 h) is increased in obese ZDF rats by pair feeding and intermittent stress but to a significantly lesser degree by intermittent stress. B, Intermittent restraint stress, but not pair feeding, reduces POMC mRNA expression in the anterior pituitary. Data are presented as mean ± SEM; n = 4 (Ln-FWR control) to 10 (obese groups) rats per group for ACTH; n = 7–10 rats per group for POMC mRNA in situ hybridization. *, P < 0.05 vs. Ln-FWR control’ {dagger}, P < 0.06 vs. Ob-FWR control; §, P ≤ 0.05 vs. Ob-FWR pair fed. AL, Anterior lobe; IL, intermediate lobe; SP, superior lobe.

 
Basal anterior pituitary POMC mRNA expression was not increased in obese control relative to lean rats but was reduced in obese rats 20% by intermittent stress (P < 0.06 and P < 0.03 vs. control and pair-fed rats, respectively) (Fig. 6BGo). POMC mRNA in the intermediate pituitary was not different between groups (data not shown).

Basal catecholamines.
Basal plasma norepinephrine and epinephrine levels (supplemental Fig. 3) were sampled every 6 h over a 24 h period during wk 13 via cannulas. Using the 24-h AUC, basal norepinephrine and epinephrine were not different in obese food/water-restricted control compared with lean rats. Similarly, neither intermittent stress nor pair feeding in obese rats altered norepinephrine (Ln-FWR control, 597 ± 51; Ob-FWR control, 648 ± 108; Ob-FWR pair fed, 857 ± 47; Ob-stress, 911 ± 92 pg/ml·h) or epinephrine compared with obese control rats (Ln-FWR control, 409 ± 116; Ob-FWR control, 234 ± 46; Ob-FWR pair fed, 169 ± 28; Ob-stress, 259 ± 67 pg/ml·h).

CRH and AVP mRNA (Fig. 7Go, A and B).
Total CRH mRNA expression in the parvocellular region of the PVN (pPVN) was not altered in obese compared with lean ZDF rats. Similarly, in obese rats, neither intermittent stress nor pair feeding altered pPVN. Similarly, CRH mRNA expression in the dorsomedial pPVN was not affected by obesity or either treatment.


Figure 7
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  		FIG. 7. In situ hybridization of basal CRH mRNA (A) and AVP mRNA (B) expression in the PVN and MR mRNA expression (C) in the hippocampus and dendate gyrus after 13 wk of intermittent restraint stress. Data are presented as mean ± SEM; n = 7–10 per group. *, P < 0.05 vs. lean; {dagger}, P ≤ 0.05 vs. control; §, P ≤ 0.05 vs. pair fed.

 
AVP mRNA expression in total and pPVN were increased about 2-fold in obese control and pair-fed rats compared with lean controls (P ≤ 0.06 vs. Ln-FWR control). In contrast, intermittent stress did not increase AVP mRNA expression, and a t test revealed that total AVP mRNA levels were about 30% lower after 13 wk of intermittent stress than in pair-fed rats (P = 0.04). However, this relationship did not reach significance by one-way ANOVA. No differences in AVP mRNA levels were apparent in the supraoptic nucleus (data not shown).

GR mRNA (Table 2Go).
GR mRNA expression in the anterior pituitary, PVN, and hippocampus were not affected by obesity or by intermittent stress or pair feeding in obese rats.


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  		TABLE 2. GR mRNA expression (ROD) in the anterior pituitary, PVN, hippocampus, and dendate gyrus in ZDF rats after 13 wk of intermittent restraint stress

 
MR mRNA (Fig. 7CGo).
Hippocampal MR mRNA expression was increased in obese compared with lean control rats (factorial ANOVA, treatment P < 0.0001). In obese rats, both intermittent stress and pair feeding lowered MR mRNA levels (factorial ANOVA, treatment P < 0.0005; post hoc P < 0.05 vs. Ob-FWR control), although pair feeding lowered MR mRNA expression more so than intermittent stress (treatment P < 0.0005; post hoc P < 0.002).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously showed that intermittent restraint stress delays development of hyperglycemia in male ZDF rats but were unable to determine to what extent this delay was caused by stress-induced reductions in food intake (18). In the current study, we first show that restraint stress acutely increases glycemia but that these glycemic responses adapt over time with intermittent exposure. This is accompanied by delay of basal fed and fasting hyperglycemia produced by both stress-induced reductions in food intake and food intake-independent actions of intermittent stress. Second, we show that intermittent stress per se leads to HPA adaptations that prevent the exacerbation of hypercorticosteronemia caused by reduced food intake. Thus, it is this prevention of hypercorticosteronemia that we postulate is responsible for the stress-induced amelioration of hyperglycemia that occurs in response to intermittent stress per se independent of reductions in food intake.

The stress-induced reduction in food intake is most likely an important contributor toward the amelioration of hyperglycemia by intermittent stress, because lowering of food intake reduces hepatic glucose production (30), improves insulin sensitivity (30, 31), and increases insulin secretion (30). Similarly, we show that adaptation to intermittent stress promotes an increase in basal relative insulinemia during the light phase. This does not differ from that in pair-fed rats, suggesting that the increase in relative hyperinsulinemia is dependent on the reduction in food intake. On the other hand, the intermittent stress-induced reduction in hyperphagia may be secondary to improvements in central insulin sensitivity. Swim stress improves hypothalamic insulin and leptin sensitivity, increasing their ability to inhibit food intake (32), and hypothalamic leptin and insulin signaling improves peripheral insulin sensitivity and lowers hepatic glucose production (33, 34). However, intermittent stress induced a 20% reduction in hyperphagia without altering insulin-sensitive NPY or POMC mRNA expression in the arcuate nucleus 24 h after stress, suggesting that changes in central insulin sensitivity did not occur. Thus, the intermittent stress-induced delay of hyperglycemia is likely partially mediated by stress-induced reductions in food intake that subsequently increase basal insulinemia and improve insulin sensitivity, as has been shown in other models of food restriction (30, 31).

Other rodent studies have observed intermittent stress-induced improvements in glycemia (20, 35). However, this is the first to show that these improvements are partially independent of food intake and entirely independent of body weight. The gradual food restriction of about 5% by 3 wk, increasing to 20% by 13 wk, delayed development of hyperglycemia without reducing body weight. The inconsistency between reduced food intake but not body weight with intermittent stress and pair feeding suggests that there is a decrease in energy expenditure and/or caloric absorption with both treatments. This is further suggested by the higher rate of weight gain normalized to food intake in the latter weeks of the study and the lack of a reduction in epididymal fat mass by either pair feeding or intermittent stress in the face of reduced food intake. Indeed, energy restriction reduces energy expenditure (36), and stress can increase fat deposition (37). Second, the gradual food restriction used in this study and caused by intermittent stress amounted to a 12–15% reduction in cumulative food intake over 13 wk, which is relatively mild in comparison with most studies using 40–50% restriction throughout the entire duration of the study. Third, obese diabetic control rats were hyperglycemic for the last 6 wk of the study and thus would have lost significant calories in the urine that would adversely affect their normal weight gain. Indeed, obese diabetic Zucker rats with 3-fold higher plasma glucose levels lose 9-fold more glucose in their urine than nondiabetic obese Zucker rats (38). This would counteract normal weight gain of the obese diabetic control rats and thus mask any effects of the gradual food restriction on body weight. We postulate that a comparison with a nondiabetic obese ZDF group would reveal reductions in body weight with food restriction. It will be important for future studies to examine total lean and fat mass and energy expenditure.

We hypothesized that the delay of hyperglycemia by intermittent stress per se is related to amelioration of the hypercorticosteronemia. Thus, we examined regulation of the HPA axis after 13 wk of intermittent stress in the obese ZDF rat. Although it is known that the Zucker fatty rat demonstrates HPA axis hyperactivity (39, 40, 41), to our knowledge, our study is the first to examine the HPA axis in ZDF rats, which, in contrast to Zucker fatty rats, develop diabetes. Thus, we describe the effect of diabetes, pair feeding, and intermittent stress per se on the HPA axis. Obese ZDF rats demonstrated basal hypercorticosteronemia, increased corticosterone responses to stress (food/water removal), and increased basal AVP mRNA expression in parvocellular neurons of the PVN. The increased AVP but not CRH mRNA levels are likely due to the approximately 6 wk of hyperglycemia preceding brain removal, because AVP increases during chronic stress (11, 42). Elevated AVP occurred despite increased hippocampal MR mRNA, similar to in streptozotocin-diabetic rats (43, 44) that have increased CRH mRNA (43). This suggests diabetes increases central drive on the HPA axis. We hypothesize this increased central drive is related to hyperglycemia.

Food restriction increases basal corticosteronemia in normal rats (45). We show this occurs even in hypercorticosteronemic ZDF rats and may be independent of ACTH, similar to normal rats (45). The increased nadir corticosterone to ACTH ratio is consistent with increased in vivo adrenal responses to ACTH in chronically food-restricted normal rats (46). However, because ACTH secretion is extremely pulsatile (47), this interpretation is cautiously made. Pair feeding reduced hippocampal MR mRNA expression, suggesting reduced hippocampal suppression of HPA activity. Despite this, PVN CRH and AVP mRNA were not increased, suggesting amelioration of central drive on the HPA axis, consistent with our hypothesis that this heightened central drive is related to hyperglycemia.

The effect of intermittent stress on the HPA axis in diabetic rats that demonstrate HPA hyperactivity is poorly understood and is important because hypercortisolemia occurs in T2DM patients and is related to poor metabolic control (48). The reduction in food intake, marked initial corticosterone responses to restraint, and previously demonstrated inhibition of GH (18) are evidence of HPA activation and a stress response. However, it deserves mention that intermittent stress did not cause adrenal hypertrophy or atrophy of the thymus, indices of chronic stress dependent on corticosterone hypersecretion. Similarly, adrenal hypertrophy in streptozotocin-diabetic rats is not exacerbated with recurrent restraint (49), and forced swim stress also does not induce adrenal hypertrophy in ZDF rats (19), consistent with our results.

Although we previously observed normalized basal corticosterone levels after 13 wk of intermittent stress (18), a similar reduction was not observed in this study using multiple basal corticosterone measurements over 24 h. Not surprisingly, corticosterone levels in response to restraint stress habituated over time. These levels became lower than those induced by intermittent exposure to the less severe stressor of 1 h food/water removal in obese ZDF control rats and became comparable to levels induced by food/water removal in lean ZDF rats. This was associated with lower glycemic responses to 1 h restraint stress compared with food/water restriction, which may be clinically relevant because glycemic excursions before diabetes can increase risk of diabetic complications (50). Despite reducing hippocampal MR mRNA levels after 13 wk, intermittent stress showed a trend to reduce AVP and POMC mRNA levels and significantly lowered stress-induced corticosterone levels. Thus, adaptation to intermittent stress ameliorates HPA activity, possibly through reduced central drive on the HPA axis.

Most importantly, substantial adaptations in the HPA axis with intermittent restraint occurred independent of food intake. Intermittent restraint prevented the basal hypercorticosteronemia caused by pair feeding. This was accompanied by habituation of the corticosterone response to restraint stress, reaching lower levels than those induced by food/water removal in obese intermittently food/water-restricted pair-fed rats. These adaptations in corticosterone likely occurred via increased hippocampal MR mRNA, lower POMC mRNA in the anterior pituitary, and lower adrenal sensitivity to ACTH as is suggested by the lower ratio of ACTH to corticosterone in intermittently stressed rats during the corticosterone nadir. Thus, these adaptations may explain the food intake-independent delay of hyperglycemia with intermittent stress. Fasting glycemia is reduced 40% upon reduction of hepatic and adipose GR expression in ZDF rats (51), demonstrating that reduced glucocorticoid exposure can improve glycemia in ZDF rats.

Our speculations at this time regarding molecular regulation of the HPA axis are based exclusively upon mRNA expression. We previously demonstrated strong correlations between hippocampal MR mRNA (r = 0.89; P < 0.0001) and GR mRNA (r = 0.67; P < 0.001) and protein expression (43). However, future studies should address whether similar changes are reflected at the level of protein expression.

Chronic stress in rodents can increase ingestion of palatable/high-energy foods (52), which reduces HPA activity and has been suggested to be a compensatory behavioral response to prevent HPA axis hyperactivity from chronic stress (52). We show that similar to ingestion of palatable foods, intermittent stress reduces basal HPA hyperactivity associated with the food restriction, which further ameliorates hyperglycemia. Thus, future directions for these studies should examine whether similar benefits in terms of HPA activity and glycemia occur when rats are given a high-energy food choice, similar to the food choices available to humans.

In conclusion, we propose that intermittent restraint stress delays hyperglycemia in the male ZDF rat partially through stress-induced reductions in food intake that likely improve insulin sensitivity (30, 31) and maintain β-cell compensation (18, 30) and that intermittent stress also ameliorates hyperglycemia independent of reductions in food intake through adaptations that reduce exposure to the hyperglycemic effects of corticosterone (Fig. 8Go). This may be mediated by up-regulation of hippocampal MR levels to increase suppression of HPA activity and/or lowering adrenal sensitivity to ACTH, thus preventing the worsening of hypercorticosteronemia seen with food restriction. Glucocorticoid excess occurs in T2DM patients and is associated with increased severity of diabetes and greater risk of diabetic complications (48). We demonstrate that food restriction, a common preventative/treatment strategy for early diabetes, is a mild stressor and worsens the glucocorticoid excess associated with diabetes. In contrast, adaptation to intermittent psychogenic stress prevents this worsening of glucocorticoid levels while simultaneously lowering food intake and thus ameliorates hyperglycemia better than food restriction alone. Importantly, this contrasts with common views that all stressors are deleterious for diabetes and illustrates that intermittent exposure to stressors and the ensuing adaptations may instead be important for normal physiological functioning by preparing the body to deal with threats to homeostasis.


Figure 8
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  		FIG. 8. Current model for mechanism of stress-induced amelioration of hyperglycemia. A, Reduced food intake alone (pair feeding) ameliorates hyperglycemia, but this is counteracted by the hyperglycemic effects of increased adrenal secretion of nadir corticosterone. B, Intermittent stress ameliorates hyperglycemia through reducing food intake but further improves glycemia by preventing the increase in adrenal sensitivity and increasing hippocampal glucocorticoid feedback, thus preventing the increase in nadir corticosterone.

 


   Acknowledgments
 
We thank Elena Burdett for her technical support.


   Footnotes
 
Please address reprint requests to Dr. Mladen Vranic, Department of Physiology, Medical Sciences Building, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: mladen.vranic{at}utoronto.ca.

This work was supported by a research grant to M.V. and S.G.M. from the Canadian Institutes of Health Research (CIHR) (MOP-2197). H.B. was supported by CIHR Canada Graduate Scholarship Doctoral Award (CGD-76341). M.K. is a recipient of a Natural Sciences and Engineering Research Council of Canada Doctoral Award and Banting and Best Diabetes Centre (BBDC) Novo-Nordisk Scholarship, and J.Y. is a recipient of a CIHR Doctoral Award, and a BBDC Novo-Nordisk Scholarship.

This study was presented in part in poster form at the 67th American Diabetes Association Scientific Sessions held in Chicago, Illinois, June 22–26, 2007.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2008

1 M.V. and S.G.M. contributed equally as senior authors. Back

Abbreviations: AUC, Area under the curve; AVP, arginine vasopressin; FWR, food/water restriction; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; MR, mineralocorticoid receptor; Ob, obese; POMC, proopiomelanocortin; pPVN, parvocellular region of the PVN; PVN, paraventricular nucleus; ROD, relative OD; T2DM, type 2 diabetes mellitus; ZDF, Zucker diabetic fatty.

Received October 26, 2007.

Accepted for publication February 26, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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