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Endocrinology, doi:10.1210/en.2003-1748
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Endocrinology Vol. 145, No. 9 4309-4319
Copyright © 2004 by The Endocrine Society

Pyruvate Prevents Restraint-Induced Immunosuppression via Alterations in Glucocorticoid Responses

Gretchen N. Neigh, Stephanie L. Bowers, Leah M. Pyter, Michelle L. Gatien and Randy J. Nelson

Departments of Psychology and Neuroscience, The Ohio State University, Columbus, Ohio 43210

Address all correspondence and requests for reprints to: Randy J. Nelson, Townshend Hall, 1885 Neil Avenue Mall, Columbus, Ohio 43210. E-mail: rnelson{at}osu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stress-evoked immunosuppression may reflect increased demands on cellular energy signaled via elevated glucocorticoid concentrations. We hypothesized that treatment with pyruvate, an alternative energy source, would ameliorate restraint-induced elevation of glucocorticoids and that this reduction in glucocorticoid exposure will prevent stress-induced immunosuppression. We provided exogenous pyruvate to mice exposed to repeated restraint and then assessed splenocyte counts and splenocyte proliferation in response to the mitogen, concanavalin A as well as IgM production in response to keyhole limpet hemocyanin. Immune function was suppressed in mice undergoing repeated restraint but not in mice exposed to repeated restraint followed by pyruvate treatment. All mice exposed to restraint, regardless of pyruvate supplementation, displayed equivalent occurrences of repeated elevations in corticosterone concentrations; however, the cumulative exposure to corticosterone after one episode of restraint was reduced in those mice treated with pyruvate after restraint. Finally, we tested the immunoprotective ability of pyruvate supplementation in the presence of chronically elevated corticosterone. Mice implanted with restraint-like concentrations of corticosterone after adrenalectomy decreased splenocyte counts, compared with either unmanipulated mice or mice that were implanted with a cholesterol pellet after adrenalectomy, regardless of pyruvate supplementation. These data suggest that pyruvate does not possess immunoprotective properties in the presence of chronically elevated corticosterone. Pyruvate supplementation preserves immune function during exposure to repeated restraint stressors; altered dynamics of corticosterone concentrations after pyruvate administration may mediate this immunoprotection. Pyruvate prevents restraint-induced immunosuppression via alterations in the glucocorticoid response to restraint.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE TO REPEATED stressors can cause or intensify some of the most common human ailments, including heart disease, cancer, irritable bowel syndrome, and depression (1). Stress-evoked disorders may result from a chronic disturbance in homeostasis termed allostatic load (2). To restore homeostasis, behavioral and physiological adjustments must match the available energy of the organism to the energetic demands. Common adjustments include reduced sexual behavior (e.g. see Ref.3), stunted growth (4), or compromised immune function (e.g. see Ref.5).

Exogenous glucocorticoids can exert immunosuppressive effects, and high concentrations of endogenous glucocorticoids compromise immune function (see Ref.6). However, a recent study demonstrated that immunosuppression is not obligatory after repeated elevations in glucocorticoid concentrations. Supplementing mice with pyruvate prevents restraint-induced suppression of mitogen-stimulated splenocyte proliferation despite repeated elevations in corticosterone concentrations (7). Because the glucocorticoid response mobilizes stored energy, if pyruvate meets the energetic need created by the stressor, then pyruvate supplementation may expedite the return of glucocorticoid concentrations to basal concentrations. Minimizing the duration of each glucocorticoid response, despite a similar number of elevations in corticosterone concentrations, may preserve immune function by decreasing the total cumulative exposure to corticosterone.

Supplementation with pyruvate has the potential to directly provide sufficient ATP and nicotinamide adenine dinucleotide to restore homeostasis; however, this seems unlikely due to the efficacy of a comparatively small dose (0.5 mg/kg) to prevent stress-evoked immunosuppression (7). Alternatively, the immunoprotective effects of pyruvate may depend on, or directly relate to, the potential effects of pyruvate on corticosterone and other hormones involved in energetic signaling. Hormones such as glucocorticoids, insulin, and leptin interact to mediate energy balance (8, 9, 10), and therefore pyruvate may act by affecting a combination of these hormones. Bolus administration of pyruvate alters both pancreas and liver function (11, 12), suggesting that either insulin or fatty acid signaling may mediate the effects of pyruvate. Insulin decreases after exposure to chronic stressors (13), and elevated insulin can attenuate corticosterone concentrations (14). Importantly, pyruvate increases insulin secretion under some conditions (11). Additionally, the release of fatty acids alters leptin signaling to the central nervous system, a component of the glucocorticoid-metabolic-brain feedback axis (15). Exposure to acute stress and glucocorticoids promote lipolysis from adipose tissue, thereby increasing free fatty acids (16, 17) that may account for stress-induced alterations in leptin signaling. Rats exposed to restraint for 10 d decrease leptin concentrations, and peripheral infusion of leptin suppresses restraint-induced corticosterone and CRH secretion (18, 19). Furthermore, pyruvate stimulates the synthesis and release of leptin from cultured adipocytes (20). Therefore, we hypothesize that an interaction among corticosterone, insulin, and leptin mediates the immunoprotective effects of pyruvate by signaling the preservation of energy balance. Furthermore, we suggest that the immunoprotective effects of pyruvate supplementation are dependent on alterations in glucocorticoid concentrations.

The data presented here confirm that mice exposed to restraint followed by pyruvate supplementation maintain immune function. Analysis of the corticosterone response to restraint suggests that pyruvate diminishes the total exposure to increased corticosterone concentrations and may do so through alterations in either insulin or leptin signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The Institutional Laboratory Animal Care and Use Committee of Ohio State University approved all animal procedures in accordance with the guidelines set forth by the National Institutes of Health. We housed adult male C57BL/6 mice (12 wk of age) individually in polycarbonate cages (28 x 17 x 12 cm) from the onset of the study in rooms programmed to be illuminated for 14-h light, 10-h dark (lights illuminated at 2400 h Eastern Standard Time) and maintained at temperatures of 20 ± 4 C and relative humidity of 50 ± 5%. Tap water was available ad libitum throughout the study. This study consisted of four experiments.

Experiment 1: effects of pyruvate on immune function
Mice were randomly assigned to one of seven experimental groups: 1) handled (n = 12), 2) saline (n = 12), 3) 0.5 mg/kg pyruvate (n = 12), 4) 5.0 mg/kg pyruvate (n = 12), 5) restraint + saline (n = 10), 6) restraint + 0.5 mg/kg pyruvate (n = 8), and 7) restraint + 5.0 mg/kg pyruvate (n = 12). The experiment was conducted in three iterations with all experimental groups represented in each replication. Some environmental perturbations occurred in the colony room during the first run of the experiment. Mice that had corticosterone concentrations that deviated by more than 3 SEM from the remainder of the group run during the second and third blocks were excluded from further analysis.

Food (LabDiet 5001; PMI Nutrition, Brentwood, MO) intake was monitored beginning 10 d before the start of the experiment and continued for the duration of the experiment for animals exposed to restraint + saline. During the experiment, we used the food intake of the group that received daily restraint + saline to determine the food provisions allotted to the remaining groups, such that each animal received the same percentage of their baseline food intake that corresponded to the average percentage basal intake that the restraint + saline group consumed. We used this method of food allotment to control for possible effects of restraint or pyruvate administration on food intake, thereby controlling for energy intake.

Restraint
Mice were placed in adequately ventilated clear polypropylene restrainers (50-ml conical tubes measuring 9.7 cm in length and internal diameter of 2.8 cm) for 3 h/d; breathing was monitored for all mice to make certain animals were not compressed. Animals were subjected to randomly timed (between 0700 and 1200 h) restraint once daily for 6 wk.

Pyruvate injections
Sodium pyruvate (0.5, 5.0 mg/kg; Sigma Chemical Co., St. Louis, MO) was dissolved in sterile water and pyruvate was administered by ip injection immediately after each episode of restraint in all three experiments (i.e. upon removal from the restraint tube). We injected an additional group of mice ip with osmolarity-matched sodium chloride solution immediately after removal from the restraint tube. Groups not exposed to restraint received either a saline or sodium pyruvate (0.5 or 5.0 mg/kg) injection at the same time of day as the animals exposed to restraint. Additionally, to control for the effects of an injection, one group was handled each day.

Splenocyte counts and proliferation
Spleens were aseptically removed and splenocytes were assessed for proliferation in response to the mitogen concanavalin A (ConA; Sigma) 24 h after the final treatment on d 43. Splenocyte proliferation was assessed using a colorimetric assay based on the tetrazolium salt 3-(4,5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-suofophenyl)-2H-tetrazolium (Promega, Madison, WI). Splenocytes were separated from the tissue by compressing the whole spleen between sterile frosted glass slides. The resulting slurry was suspended in 3 ml RPMI 1640 (Sigma) and layered onto 2 ml Ficoll (Sigma). Tubes were spun at 2500 rpm for 30 min, and then the white blood cell layer was removed, placed in a sterile tube, and spun for 10 min at 1500 rpm. The resulting pellet was suspended in 200 µl supplemented culture medium (RPMI 1640/HEPES supplemented with 1% penicillin (5000 U/ml)/streptomycin (5000 µl/ml), 1% glutamine (2 mM/ml), 0.1% 2-mercaptoethanol (5 x 10–2 M/ml) and 10% heat-inactivated fetal bovine serum).

Splenocyte counts and viability were determined with a hemacytometer and trypan blue exclusion. Viable cells were adjusted to 2 x 106 cells/ml by dilution with culture medium, and 50 µl aliquots of each cell suspension (i.e. 100,000 cells) were added to the wells of sterile culture plates. ConA was diluted with culture medium to concentrations of 10 µg/ml, a concentration previously determined to be optimal for stimulation of mouse splenocytes. Plates were then incubated at 37 C with 5% CO2 for 48 h. Plates were removed from the incubator, 20 µl 3-(4,5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-suofophenyl)-2H-tetrazolium solution were added to each well, and then the plates were incubated at 37 C with 5% CO2 for an additional 4 h. The OD of each well was determined with a microplate reader (490 nm; Benchmark; Bio-Rad Laboratories, Hercules, CA). In subsequent statistical analyses, mean OD values were used for each set of duplicates.

Humoral immune function
To assess humoral immunity, after the 11th exposure to restraint, mice were injected sc with the antigen, keyhole limpet hemocyanin (KLH; 150 µg suspended in 0.1 ml sterile 0.9% saline), to which they were previously naïve. KLH evokes an acute immune response but does not replicate or cause long-term fever or inflammation (21). IgM specific for KLH was measured in a blood sample obtained 15 d before immunization and on d 3, 10, 17, 24, and 32 post immunization using a specific ELISA. Diluted serum samples (1:100) were added to antigen-coated plates that were previously blocked with 0.5% milk in PBS to reduce nonspecific binding. After incubation, secondary antibody (AP-conjugated antimouse IgM, 1:1000) was added, and after further incubation, the enzyme substrate p-nitrophenyl phosphate was added. The absorbance of each well was determined using a Bio-Rad plate reader (405 nm). Mean absorbance for each sample was expressed as a percent plate-positive control for statistical analyses.

Pyruvate and lactate assay
On d 43, trunk blood was collected to assess the effect of chronic restraint on metabolic function. Pyruvate and lactate concentrations, as well as the pyruvate/lactate ratio, were determined. Because pyruvate is converted to lactate during anaerobic metabolism and this pathway produces fewer ATP per unit of pyruvate than the aerobic pathway, assessing the pyruvate/lactate ratio provided an index of metabolic efficiency. Enzymatic determination kits (Sigma Diagnostics, St. Louis, MO) were used to quantify the amount of pyruvate and lactate present in the blood.

Corticosterone RIA
All blood samples were obtained from the retroorbital sinus under light isofluorane anesthesia, and serum corticosterone was determined by RIA. Serum was collected within 60 sec of disturbing the animal. This is an adequately short period of time to allow for the collection of basal corticosterone concentrations (22). We conducted the RIA following the guidelines in the ICN Biochemicals (Costa Mesa, CA) 125I double-antibody kit instructions. The RIA is highly specific, with a cross-reaction of less than 1% with other hormones and a detection limit of 5 ng/ml. The coefficients of variation were less than 10%, and the intraassay variation was less than 4%. Corticosterone concentrations were measured from blood collected 4 d before experiment onset and on d 1, 7, 14, 21, 28, 35, 42, and 43 to assess the effect of each treatment. Blood samples were collected 10 min post treatment.

Experiment 2: endocrine response to acute restraint
Mice were randomly assigned to one of five experimental groups (n = 10 per group): 1) handled, 2) saline, 3) 0.5 mg/kg pyruvate, 4) restraint + saline, and 5) restraint + 0.5 mg/kg pyruvate. In addition, the effect of each treatment condition was assessed at three time points: 5, 30, or 60 min post treatment. Mice were not sampled repeatedly; a separate cohort of mice was sampled at each time point for each treatment condition. Experiment 2 was conducted in three iterations with all experimental groups represented in each replication.

Mice in experiment 2 had ad libitum food until the day of treatment. On the day of treatment, food was removed immediately after treatment until the time of sample collection. Animals were restrained as in experiment 1 except that mice in the restraint groups were subjected to only one episode of restraint (0800–1100 h). Corticosterone concentrations were determined from blood collected 4 d before the experiment and then 5, 30, or 60 min after the termination of treatment. Insulin and leptin concentrations were determined from trunk blood collected 5, 30, or 60 min after the termination of treatment.

Area under the curve (AUC)
Corticosterone concentrations were determined as described in experiment 1 and graphed as lines without symbols, and ImageJ software (version 1.31) was used to determine the AUC. The methods used have been previously described (23).

Insulin RIA
The insulin RIA was performed following the guidelines in the ICN Biochemicals 125I-coated tube kit instructions. The RIA is highly specific, with a cross-reaction of less than 1% with other hormones and a detection limit of 5.5 µIU/ml. The coefficients of variation were less than 15%.

Leptin RIA
The leptin RIA was performed following the guidelines in the Linco Research, Inc. (St. Charles, MO) 125I mouse leptin RIA kit instructions. The RIA is highly specific, with a cross-reaction of less than 1% with other hormones and a detection limit of 0.2 ng/ml. The coefficients of variation were less than 10%.

Experiment 3: endocrine response to chronic restraint
Mice were randomly assigned to one of five experimental groups: 1) handled, 2) saline, 3) 0.5 mg/kg pyruvate, 4) restraint + saline, and 5) restraint + 0.5 mg/kg pyruvate. Two iterations of experiment 3 were conducted with all treatment groups represented in each iteration. Corticosterone, insulin, and leptin concentrations were determined only from mice from the first iteration of the experiment (n = 11 per group). Mice in the second phase of the experiment (n = 8 per group) were prepared for immunohistochemical analysis of CRH.

Mice in experiment 3 had ad libitum food except on the days of sample collection. On sample collection days, food was removed immediately after treatment until sample collection 60 min later. Food intake was measured daily, and there were no significant pretreatment differences in food intake among the groups. Animals were restrained as in experiment 1 except that mice were restrained for 3 h daily for 3 wk. Corticosterone concentrations were determined from blood collected 4 d before the experiment and 60 min post treatment on d 1, 6, and 20. Insulin and leptin concentrations were measured from trunk blood collected 60 min post treatment after the 20th exposure to restraint.

Immunohistochemistry
After 3 wk of exposure to the assigned treatment, mice were returned to their home cages for 60 min. Mice were injected with an overdose of sodium pentobarbital and transcardially perfused with 0.9% saline (4 C) followed by 4% paraformaldehyde (4 C). Brains were removed, placed in 4% paraformaldehyde for 24 h at 4 C, and then transferred into 30% sucrose in PBS solution for 24 h at 4 C. Brains were frozen on dry ice and stored at –80 C until processing. Brains were sectioned into 30-µm sections on a cryostat and thaw mounted onto slides. Slides were rinsed in PBS (pH 7.2) and then placed in anti-CRH antibody (diluted 1:2000 in PBS; courtesy of Dr. W. Vale, The Salk Institute, La Jolla, CA) for 72 h at 4 C on a rotator. Slides were again rinsed in PBS and then placed in cy3 secondary antibody (1:2000 in PBS; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature on a rotator. Slides were again rinsed in PBS and then coverslipped with immunomount (Thermo Shandon, Pittsburgh, PA). Phase-contrast microscopy was used to identify the coronal sections that contained the paraventricular nucleus of the hypothalamus (PVN; bregma –0.58). Photomicrographs were captured at x400 magnification, and the exposure was automatically adjusted to normalize the intensity of the background. A standard box was drawn in the center of the PVN, as demonstrated in Fig. 7AGo, and CRH expression was semiquantitatively assessed with Inquiry Autoradiography software (Loates Associates, Westminster, MD). Median OD was determined from three to four sections containing the PVN, and then the median value for each group was calculated.



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  		FIG. 7. A, A schematic drawing of the approximate coronal level used for analysis of CRH in the PVN [adapted from Paxinos and Watson (45 )]. The inset depicts an enlarged representation of the area of interest. The gray box corresponds to the region analyzed for CRH protein and corresponds to the photomicrographs presented in C–E. B, This box plot demonstrates the median and range of data recorded from the region illustrated in A for mice that were handled, exposed to 3 h of restraint followed by a saline injection (restraint + sal), or exposed to 3 h of restraint followed by a 0.5 mg/kg pyruvate (PYR) injection (restraint + PYR) for 3 wk. C, A representative photomicrograph (x400) of CRH in the PVN of mice exposed to 3 wk of daily handling. D, CRH expression is reduced in the PVN of mice exposed to 3 wk of restraint + saline injections, compared with the handled mice. E, Mice in the restraint + 0.5 mg/kg pyruvate group do not exhibit a reduction in CRH in the PVN, compared with the handled mice.

 
Experiment 4: immunoprotective effect of pyruvate in the presence of elevated corticosterone
Mice were randomly assigned to one of five experimental groups: 1) no surgery, 2) adrenalectomy (ADX) + cholesterol implant + saline, 3) ADX + cholesterol implant + 0.5 mg/kg pyruvate, 4) ADX + corticosterone implant + saline, and 5) ADX + corticosterone implant + 0.5 mg/kg pyruvate. After 1 wk of habituation to the colony, mice were anesthetized with isofluorane and underwent bilateral adrenalectomy and were sc implanted with the designated pellet. Mice were provided with separate bottles containing tap water and isotonic saline for the duration of the study. In addition to standard lab chow, mice were provided with mash made with isotonic saline daily.

Twenty-four hours after ADX surgery, we began daily injections as dictated by their treatment groups. After 14 d of treatment, a duration known to produce immunosuppression (7), trunk blood was collected for analysis of corticosterone concentrations, and spleens were aseptically removed for determination of total splenocytes and splenocyte proliferation in response to the mitogen ConA, as described in experiment 1.

Data analyses and statistics
Splenocyte proliferation in response to 10 µg ConA was expressed as mean OD. In experiment 1, two data points (one from the restraint + saline and one from the restraint + 0.5 mg/kg pyruvate) were more than 2 SD away from the mean and were excluded from further analysis. The remaining data were log transformed. KLH, pyruvate, and lactate values were calculated as the percent plate positive and then averaged within each group. Splenocyte counts, corticosterone, insulin, and leptin concentrations were averaged within each group. Splenocyte counts, splenocyte proliferation, basal corticosterone concentration in experiments 2 and 4, and insulin and leptin concentrations in experiment 3 were analyzed with a one-way ANOVA. If the assumption of normality was violated, then a Kruskal-Wallis ANOVA on ranks was substituted. To analyze effects of treatment and time, corticosterone, insulin, and leptin concentrations in experiment 2 were assessed with a two-way ANOVA. Two-way repeated-measures ANOVAs were used to assess differences of treatment and time for humoral immune function and corticosterone concentrations in experiment 1 as well as for corticosterone concentration in experiment 3. The Tukey pairwise comparison or Dunn’s test were used to further distinguish among groups, and all differences were considered statistically significant if P < 0.05 unless the assumptions of normality or equal variance were violated. If these assumptions were violated, then we adjusted to P < 0.025 for initial analyses and P < 0.01 for post hoc analyses to correct for the increased likelihood of type I error (24). Mann-Whitney rank sum tests were used for the analysis of the semiquantitative measure of CRH protein in the PVN.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: effects of pyruvate on immune function
Splenocyte counts and proliferation.
Repeated restraint (n = 10) reduced the total number of splenocytes and suppressed splenocyte proliferation in response to 10 µg ConA, compared with mice exposed to daily handling (n = 12; P < 0.05). Pyruvate supplementation (0.5 or 5.0 mg/kg; n = 8 and n = 10, respectively) prevented this suppression (P > 0.05, compared with handled mice; Fig. 1BGo). Neither saline injections (n = 12) nor pyruvate injections (0.5 mg/kg, n = 12, or 5.0 mg/kg, n = 12) exerted suppressive effects on splenocyte counts or splenocyte proliferation (P > 0.05; Fig. 1AGo).



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  		FIG. 1. A, Splenocyte counts are suppressed after 6 wk of restraint, compared with counts from mice that underwent 6 wk of restraint in combination with daily pyruvate (PYR) supplementation (0.5 mg/kg; &, P < 0.05). B, Splenocyte proliferation to the mitogen ConA is suppressed after 6 wk of restraint, compared with handled mice (*, P < 0.05). Pyruvate supplementation after restraint (0.5 or 5.0 mg/kg) prevents restraint-induced suppression of splenocyte proliferation. Neither saline injections nor pyruvate injections (0.5 or 5.0 mg/kg) exerted suppressive effects on splenocyte proliferation.

 
Humoral immune function.
Mice exposed to repeated restraint (n = 10) produced fewer anti-KLH IgM antibodies, compared with handled mice (n = 12; P < 0.05; Fig. 2BGo). Pyruvate supplementation (0.5 mg/kg; n = 8) prevented the restraint-induced suppression of anti-KLH IgM antibodies (P < 0.05, compared with restraint + saline; P > 0.05, compared with handled mice); however, this effect was dose specific such that the higher dose of pyruvate (5.0 mg/kg; n = 12) was not effective (P > 0.05, compared with restraint + saline; P < 0.05, compared with handled). In the absence of restraint, there was no significant effect of either saline or low-dose pyruvate (0.5 mg/kg) treatment (P > 0.05, compared with handled mice); however, the higher dose of pyruvate supplementation (5.0 mg/kg) suppressed anti-KLH IgM production, compared with handled mice (P < 0.05; Fig. 2AGo).



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  		FIG. 2. Mice were injected with KLH (150 µg) after the 11th exposure to their respective treatment conditions. Anti-KLH IgM was measured in serum obtained at multiple time points after immunization. A, The low dose of pyruvate (0.5 mg/kg) did not exert any effects on anti-KLH IgM production independent of restraint; however, the higher dose of pyruvate (5.0 mg/kg) suppressed the production of anti-KLH IgM, compared with handled mice. B, Restraint resulted in a reduction of anti-KLH IgM, compared with handled mice; however, low-dose pyruvate supplementation (0.5 mg/kg) during restraint prevented this suppression such that anti-KLH IgM concentrations were not different from those exhibited by handled mice. The higher dose of pyruvate (5.0 mg/kg) was not protective such that mice treated with 5.0 mg/kg pyruvate during the restraint paradigm exhibited suppressed anti-KLH IgM concentrations, compared with handled mice.

 
Corticosterone.
All treatment groups displayed similar basal corticosterone concentrations before the onset of treatment (P > 0.05; Fig. 3Go). Mice injected with either saline or 5.0 mg/kg pyruvate had elevated corticosterone concentrations, compared with handled mice; however, mice injected with 0.5 mg/kg pyruvate did not increase corticosterone. All restraint treatments elevated corticosterone, compared with the nonrestrained groups (P < 0.05). There were no differences in the magnitude of the corticosterone elevation between the restraint conditions (P > 0.05).



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  		FIG. 3. Serum was analyzed for corticosterone concentrations after the first treatment (first bar in each group) and then at 1-wk intervals throughout the 6 wk (each subsequent bar in each group). All restraint conditions produced elevations in serum corticosterone, compared with either baseline samples or handled mice. There were no differences in the number of increases or the corticosterone concentrations among the groups exposed to restraint, regardless of pyruvate (PYR) treatment.

 
Pyruvate and lactate.
Twenty-four hours after the final treatment, there were no differences in pyruvate concentrations among the treatment groups. However, median lactate concentrations were increased in the mice exposed to restraint and then treated with pyruvate (0.5 or 5.0 mg/kg; P < 0.001).

Experiment 2: time course of acute endocrine response to restraint
Corticosterone.
All treatment groups exhibited similar basal corticosterone concentrations before the onset of treatment. Handling, saline injection, or pyruvate injection (0.5 mg/kg) did not affect corticosterone concentrations 5, 30, or 60 min post treatment (P > 0.05; Fig. 4AGo). Restraint altered corticosterone concentrations differentially, depending on drug treatment. Exposure to restraint + saline elevated corticosterone concentrations 5, 30, and 60 min post treatment, compared with either handled or saline-treated mice (P < 0.05). Exposure to restraint + 0.5 mg/kg pyruvate elevated corticosterone concentrations 5 min after treatment (P < 0.05) but not 30 or 60 min after the treatment, compared with either basal values (P > 0.05) or handled, saline, or 0.5 mg/kg pyruvate-treated mice (P > 0.05). The amplitude of the corticosterone response to restraint did not differ between mice treated with saline or pyruvate (P > 0.05). However, by 60 min post treatment, mice exposed to restraint + 0.5 mg/kg pyruvate had significantly lower serum corticosterone concentrations than mice exposed to restraint + saline (P < 0.05).



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  		FIG. 4. Upper panel, Corticosterone concentrations were sampled 5, 30, or 60 min after the termination of restraint and administration of either saline or pyruvate (PYR; 0.5 mg/kg). Restraint elevated serum corticosterone, compared with baseline concentrations (*, P < 0.05, compared with handled mice). Corticosterone concentrations of mice treated with pyruvate after restraint returned to basal values before those of the mice treated with saline (&, P < 0.05, compared with restraint + saline). Lower panel, Analysis of AUC suggests that the total exposure to corticosterone is lower for mice exposed to restraint + 0.5 mg/kg pyruvate (AUC = 11294 ng/ml·min) than for mice exposed to restraint + saline (AUC = 14782 ng/ml · min).

 
Analysis of AUC suggests that the total exposure to corticosterone was lower for mice exposed to restraint + 0.5 mg/kg pyruvate (AUC = 11294 ng/ml*min) than for mice exposed to restraint + saline (AUC = 14782 ng/ml*min; Fig. 4BGo).

Insulin.
Mice exposed to restraint followed by either saline or 0.5 mg/kg pyruvate injection exhibited lower concentrations of insulin than any of the unrestrained groups (P < 0.05; Table 1Go).


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  		TABLE 1. Treatment effects on insulin and leptin concentrations

 
Leptin.
Leptin concentrations were lower in restrained mice injected with 0.5 mg/kg pyruvate, compared with mice either handled or injected with saline (P < 0.05; Table 1Go).

Experiment 3: chronic endocrine response to restraint
Corticosterone.
All treatment groups exhibited similar basal corticosterone concentrations before the onset of treatment. Mice in the restraint + saline group exhibited elevated serum concentrations of corticosterone 60 min after the termination of the stressor (P < 0.05, compared with all other groups; Fig. 5Go) on d 1, 6, and 20. Mice that received pyruvate (0.5 mg/kg) did not elevate corticosterone concentrations at this same time point on any of the days tested (P > 0.05, compared with handled mice).



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  		FIG. 5. Corticosterone concentrations were sampled 4 d before treatment and 60 min after the treatment ({blacktriangleup}, restraint + saline; {blacksquare}, restraint + 0.5 mg/kg pyruvate; {square}, 0.5 mg/kg pyruvate; {circ}, handled; {Delta}, saline) on d 1, 6, and 20. Mice in the restraint + saline group exhibited elevated corticosterone concentrations at all time points, whereas the mice treated with pyruvate had basal concentrations of corticosterone 60 min post restraint.

 
Body mass.
Percent change of body mass on the final day of the experiment was highest in the saline, restraint + saline, and restraint + 0.5 mg/kg pyruvate groups. Mice in these groups lost more body mass than handled mice (P < 0.05; Fig. 6AGo). Body mass loss did not differ between restraint groups, regardless of pyruvate treatment (P > 0.05).



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  		FIG. 6. A, Body mass was measured after 3 wk of handling, saline injections, 0.5 mg/kg pyruvate (PYR) injections, restraint + saline injections, or restraint + 0.5 mg/kg pyruvate. The percent change in body mass was greater in mice exposed to saline injections, restraint + saline, or restraint + 0.5 mg/kg pyruvate (*, P < 0.05, compared with handled mice). B, Insulin concentrations were reduced after either 3 wk of saline injections or restraint + saline injections (*, P < 0.05, compared with handled mice). Mice in the restraint + 0.5 mg/kg pyruvate treatment condition had higher concentrations of serum insulin than mice in the restraint + saline condition (&, P < 0.05). Leptin and insulin samples were obtained 60 min after the final treatment. C, Despite similar reductions in body mass, serum leptin concentrations were reduced after 3 wk of restraint + saline (*, P < 0.05, compared with handled mice) but not in mice given repeated saline injections or mice exposed to restraint + 0.5 mg/kg pyruvate.

 
Insulin.
Mice exposed to either a daily saline injection or restraint + saline reduced insulin concentrations, compared with the handled group (P < 0.05; Fig. 6BGo). In addition, mice exposed to restraint + saline reduced insulin concentrations, compared with mice exposed to restraint + 0.5 mg/kg pyruvate (P < 0.05).

Leptin.
Of the three groups with reduced body mass (saline, restraint + saline, restraint + 0.5 mg/kg pyruvate), only mice in the restraint + saline group decreased serum leptin concentrations (P < 0.05; Fig. 6CGo), compared with handled controls.

CRH.
Mice exposed to daily restraint + saline (n = 8) reduced expression of CRH in the PVN, compared with handled mice (n = 8; P < 0.05), whereas mice exposed to daily restraint + pyruvate did not reduce CRH protein (n = 8; P > 0.05; Fig. 7BGo). Photomicrographs of representative sections are presented in Fig. 7CGo.

Experiment 4: immunoprotective effect of pyruvate in the presence of elevated corticosterone
Corticosterone.
Serum corticosterone concentrations were substantially lower in mice that underwent bilateral ADX followed by implantation with a cholesterol pellet, compared with control mice that did not undergo surgery (P < 0.05). Conversely, mice that underwent bilateral ADX followed by implantation with a corticosterone pellet exhibited corticosterone concentrations that were higher than either the nonsurgical control mice or those mice that underwent ADX and were implanted with cholesterol pellets (Fig. 8AGo; P < 0.05).



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  		FIG. 8. A, Serum corticosterone concentrations in mice that underwent bilateral ADX followed by implantation with a cholesterol pellet (chol) were lower than those of either unmanipulated mice (*, P < 0.05) or mice implanted with a corticosterone pellet (cort) after bilateral ADX (&, P < 0.05). B, ADX surgery did not alter splenocyte number or splenocyte proliferation in response to 10 µg ConA, compared with unmanipulated mice; however, mice that underwent ADX and were implanted with corticosterone pellets demonstrated a reduction in total splenocytes, compared with either unmanipulated mice or those mice implanted with cholesterol pellets (*, P < 0.05). This effect was independent of treatment with either saline or 0.5 mg/kg pyruvate (PYR). C, There was no effect of corticosterone treatment on splenocyte proliferation in response to 10 µg ConA.

 
Splenocyte counts and proliferation.
ADX surgery followed by cholesterol implantation did not alter splenocyte number or splenocyte proliferation in response to 10 µg ConA, compared with control mice that did not undergo surgery (Fig. 8Go, B and C; P > 0.05). Mice that underwent ADX and were implanted with corticosterone pellets demonstrated a reduction in total splenocytes, compared with either nonsurgical control mice or those mice implanted with cholesterol pellets (P < 0.05). This effect was independent of treatment with either saline or 0.5 mg/kg pyruvate. There was no effect of corticosterone treatment on splenocyte proliferation to 10 µg ConA (P > 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pyruvate supplementation (0.5 mg/kg) reduced the total exposure to elevated corticosterone concentrations after exposure to restraint. These results suggest that reduction in corticosterone may account for the prevention of restraint-evoked immunosuppression in pyruvate-treated mice. Furthermore, mice treated with pyruvate during restraint attenuated alterations in leptin and insulin secretion as well as in CRH expression in the PVN.

Pyruvate supplementation protects cells in multiple scenarios including decreased neuronal death after transient global ischemia (25), inhibition of pancreatic islet cell death (26), and immunoprotection during repeated restraint (7). The mechanism by which this protection is afforded remains unclear. Pyruvate rapidly crosses the blood-brain barrier (25), affording the possibility of central nervous system sites of action. Alternatively, a peripheral effect is suggested by recent data from our laboratory. Interperitoneal injection of 14C-pyruvate (0.5 mg/kg) demonstrates that pyruvate is rapidly distributed throughout the organism with elevated levels present in the blood, spleen, liver, and adrenals 60 min after injection (Neigh, G. N., J. Chen, J. T. Dalton, and R. J. Nelson, unpublished observations). Clearly, additional work is necessary to determine the site of action, but current evidence indicates that even at small doses, pyruvate can be rapidly and widely distributed throughout an organism.

Previous studies suggest that both the antioxidant properties of pyruvate and the metabolic capacity of pyruvate are potential mediators of the protective effects documented. Multiple instances of pyruvate-mediated antioxidant activity have been documented including the reversal of hydrogen peroxide mediated oxidative stress (27, 28, 29). Furthermore, pulmonary apoptosis declined after infusion of pyruvate during resuscitation from severe hemorrhagic shock without an increase in tissue ATP levels (30), suggesting a nonmetabolic mode of action. In contrast, ATP levels increased after pyruvate infusion during ischemia (31). It is unlikely that an antioxidant effect would mediate the immediate change in hormone concentrations demonstrated in the present study. The effects of pyruvate on corticosterone concentrations after a single exposure to restraint (Fig. 4Go) and the absence of pyruvate mediated immunoprotection in the presence of chronically elevated corticosterone (Fig. 8AGo) suggest a metabolic mechanism of action. The alterations in CRH in the PVN after exposure to restraint plus pyruvate, compared with mice exposed to restraint plus saline (Fig. 7Go, B and C), suggest that pyruvate supplementation alters the central nervous system response to restraint, but it is not yet possible to determine whether these central changes were responsible for, or a product of, peripheral hormone changes.

Other types of metabolic supplementation alter the impact of stress. For example, sucrose ingestion mitigates the effects of exposure to stressors. During exposure to low temperatures, sucrose ingestion decreases the corticosterone response to the stressor and attenuates the reduction of both leptin and insulin concentrations (32). The effectiveness of sucrose is dependent on corticosterone concentrations in the stress range; i.e. sucrose ingestion is ineffective on hormone concentrations of rats not exposed to low temperatures (32). Sucrose consumption also normalizes CRH expression in the PVN of adrenalectomized rats (15). These effects of sucrose, a metabolic supplement, on stress-induced physiological changes and glucocorticoid-mediated alterations in the hypothalamus-pituitary-adrenal axis are similar to the effects of pyruvate supplementation documented in the present study, further supporting the possibility that pyruvate may be working via a metabolic pathway.

Our findings are consistent with the hypothesis that negative energy balance promotes stress-evoked immunosuppression. Exposure to stressors increases energy consumption (4, 33) and alters the metabolic profile of individuals to indicate negative energy balance (e.g. see Ref.10). Negative energy balance may evoke immunosuppression, e.g. administration of 2-deoxy-D-glucose, a glucose analog that inhibits cellular use of energy, compromises immune function (34, 35). It is interesting that stress may evoke its detrimental effects through negative energy balance in light of the fact that energy restriction, in terms of food restriction, is not a factor for humans in Western cultures or for the mice in this study that were fed ad libitum. However, one must consider that the total energy available to the organism at any one time and the energy available at the cellular level may not be coincident. One possible mechanism that could account for a depletion of cellular energy is the activation of poly-ADP-ribose polymerase (PARP). PARP is activated after DNA strand breaks and catalyzes the repair of DNA, a process that requires the use of both ATP and nicotinamide adenine dinucleotide. In conditions of cellular stress, there is an overactivation of PARP, and this results in a depletion of nicotinamide adenine dinucleotide and ATP within the cell. This energetic depletion can in turn lead to cell death. A previous study demonstrated that PARP knockout mice do not exhibit stress-evoked immunosuppression, suggesting that in the absence of PARP-mediated energy depletion, the immune system is intact (36).

The current work demonstrates that pyruvate preserves both humoral immune function and mitogen-stimulated splenocyte proliferation despite significant reduction of body mass. Mice exposed to restraint have free access to food with the exception of the 3 h spent in restraint each day. Although food was freely available, mice exposed to restraint in either the 6- or 3-wk experiments demonstrated a significant reduction of body mass. Interestingly, despite a similar reduction of body mass, the mice supplemented with pyruvate (0.5 mg/kg) during the restraint paradigm demonstrated an attenuated reduction of both insulin and leptin concentrations, compared with the mice given saline during the restraint paradigm. It appears that pyruvate alters the energy balance signal in a manner that maintains immune function.

Leptin, together with insulin and glucocorticoids, signals the energetic state of the organism (e.g. see Refs.8 and 37). Mice deprived of food for 48 h exhibited decreased delayed type hypersensitivity, a measure of cell-mediate immune response, by 69%; however, if food-deprived mice are injected with leptin, then the delayed type hypersensitivity response remains intact (38). In addition, Siberian hamsters exposed to short days, a condition that reduces leptin concentrations without exposure to a stressor (39, 40), suppressed humoral immune function, compared with hamsters in long-day conditions with higher leptin concentrations (39). Furthermore, leptin administration to hamsters in short days restored humoral immune function to levels similar to long-day conditions (39).

Our data suggest that reduction in the overall exposure to corticosterone via reduction in duration of a single exposure, rather than the number of exposures, mediates the immunoprotective effects of pyruvate supplementation. Furthermore, the failure of pyruvate supplementation to reverse immunosuppression induced by chronically elevated exogenous corticosterone (Fig. 8BGo) supports the hypothesis that altered dynamics of the glucocorticoid response to restraint mediate the immunoprotective effects of pyruvate. The ultimate effects of glucocorticoids are dependent on the temporal dynamics and magnitude of the exposure (41). For instance, patients with hypercortisolism due to Cushing’s disease have reduced IL-2 receptor levels; however, if similarly high concentrations of glucocorticoids are temporarily stimulated in normal individuals, then changes in IL-2 receptor activity do not occur (42). These results support the hypothesis that prolonged exposure to glucocorticoids, rather than the magnitude of a single exposure, results in immunosuppression. Conversely, glucocorticoids are necessary for a humoral immune response demonstrated by the finding that adrenalectomized rats mount an attenuated antibody response to KLH (43). Not only is the presence of glucocorticoids necessary for a humoral immune response, phasic changes in glucocorticoid concentrations are also necessary for a transition from an IgM to an IgG response (43). Furthermore, the effects of alterations in the glucocorticoid response exert physiological effects even after the resolution of the corticosterone response such that changes in corticosteroid-binding globulin may alter the effects of seemingly basal corticosterone concentrations (44), perhaps magnifying any alterations in the glucocorticoid response produced by pyruvate supplementation.

In conclusion, pyruvate prevents restraint-evoked immunosuppression and decreases the overall exposure to glucocorticoids after restraint. Repeated glucocorticoid exposure may not lead to immunosuppression if the response is efficiently resolved. A better understanding of the impact of the dynamics of glucocorticoid exposure may provide insight into understanding the mechanisms responsible for stress-evoked disorders.


   Acknowledgments
 
We thank Dr. A. Courtney DeVries for surgical instruction, Ben Korman and Jessica Meyers for technical support, Barbara Deiner-Phelan and Dr. Georgia Bishop for technical assistance, Dr. Wylie Vale for CRH antibody, and Tricia Uhor for animal care.


   Footnotes
 
This work was supported by National Institutes of Health Grants MH 57535 and MH 6614 and National Science Foundation Grant IBN00-08454.

Abbreviations: ADX, Adrenalectomy; AUC, area under the curve; ConA, concanavalin A; KLH, keyhole limpet hemocyanin; PARP, poly-ADP-ribose polymerase; PVN, paraventricular nucleus of the hypothalamus.

Received December 23, 2003.

Accepted for publication May 28, 2004.


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 Results
 Discussion
 References
 

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