The Effects of Dehydroepiandrosterone Sulfate on Counterregulatory Responses During Repeated Hypoglycemia in Conscious Normal Rats

We studied 40 male Sprague-Dawley rats (300–350 g) bred and purchased from Harlan (Indianapolis, IN). The rats were housed and individually caged under a 12:12-h light:dark cycle, with room humidity and temperature at 50–60% and 25°C, respectively. All animals had free access to water in the Vanderbilt University Animal Care Facility. All procedures for animal use were approved by the Institutional Animal Care and Use Committee at Vanderbilt University.

At 1 week before experiments, catheters were placed in the rats’ carotid artery (for blood sampling) and the external jugular vein (for infusions) under a general anesthesia mixture (5 mg/kg acepromazine, 10 mg/kg xylazine, and 50 mg/kg ketamine). Catheter lines were kept patent by flushing them with 150 unit/ml of heparin every 3 days. Rats had free access to rat diet on the days preceding surgery and the experiments. All rats used for the 2-day experiments maintained >90% of their presurgery body weight.

Four groups of rats were studied after an overnight fast during a 2-day experimental protocol, as outlined in Fig. 1. Rats were fasted overnight before each day of the 2-day study and remained conscious and unrestrained throughout the 2-day protocols. The morning of each study day, extensions were placed on the exteriorized catheters for ease of access and were removed between day-1 and -2 studies. At t = 0 min, rats were moved to an experimental cage and allowed to become acclimated to the surroundings.

Day 1 consisted of two 2-h (120–240 and 360–480 min) hyperinsulinemic-euglycemic clamps (ANTE EUG; n = 12), hyperinsulinemic- hypoglycemic clamps (ANTE HYPO; n = 12), hyperinsulinemic-euglycemic clamps plus infusion of DHEA-S (30 mg/kg) (ANTE EUG + DHEA-S; n = 8) or hyperinsulinemic-hypoglycemic clamps plus DHEA-S infusion (ANTE HYPO + DHEA-S; n = 8). The DHEA-S was continuously infused into the jugular vein starting at t = 0 min throughout day-1 procedures. DHEA-S rather than DHEA was infused because of the former’s water solubility. DHEA-S (30 mg/kg) was dissolved in 1.5 ml of normal saline and infused at a rate of 3 μl/min. This dosage of DHEA-S was chosen because it has been previously shown to have anticorticosteroid effects on the brain (22). At the conclusion of day-1 procedures, rats were fed 5–8 g of rat diet. To maintain hematocrit, after each blood draw the rats’ own erythrocytes plus normal saline were reinfused through the jugular cannula. If a rat’s hematocrit was <40% at the beginning of the day-2 studies, that rat was removed from the study. Plasma measurements of glucose were taken every 5 min during the clamp periods and at t = 240 and 480 min for insulin levels. Between morning and afternoon clamps, plasma glucose was measured every 15–30 min and glucose infusion was adjusted to maintain euglycemia at 6.1 mmol/l.

At t = 0 min, rats were moved to an experimental cage and allowed to become acclimated to the surroundings. The study consisted of a basal period (t = 90–120 min) and an experimental period (t = 120–240 min), during which time a hyperinsulinemic-hypoglycemic clamp (described below) was performed. To measure glucose kinetics during the clamp, a primed (10 μCi), constant (0.2 μCi/min) infusion of [3-³H] glucose (PerkinElmer, Boston, MA) purified by high-performance liquid chromatography (HPLC) was administered via a precalibrated infusion pump (Harvard Apparatus, South Natick, MA) at t = 0 min and continued through t = 240 min. During the experimental period, blood was drawn every 5 min for measurement of plasma glucose, every 10 min during the basal period and every 15 min during the experimental period for [3-³H]glucose, and at t = 90, 120, 180, 210, and 240 min for counterregulatory hormones. For clarity of presentation and because counterregulatory hormones were not significantly different between t = 210 and 240 min (indicating steady state), these time points were averaged in results. Rats were killed after the day-2 procedures and the placement of carotid and jugular cannulae was verified.

At t = 120–240 min (days 1 and 2) and 360–480 min (day 1 only), a primed (60 pmol · kg⁻¹ · min⁻¹) continuous (30 pmol · kg⁻¹ · min⁻¹) infusion of insulin (Eli Lilly, Indianapolis, IN) containing 9.7% (vol/vol) rat plasma was administered via a precalibrated infusion pump (Harvard Apparatus). Plasma glucose was measured every 5 min. For the euglycemic clamp, a 50% dextrose infusion was adjusted to maintain glucose at ∼6.1 mmol/l. For the hypoglycemic clamp, glucose levels were allowed to fall and a 20% dextrose infusion was adjusted to maintain glucose at ∼2.9 mmol/l for 90 min.

The rate of glucose appearance (R_a), EGP, and glucose utilization was calculated according to the methods of Wall et al. (33). EGP was calculated by determining the total R_a, which comprises both EGP and any exogenous glucose infused to maintain the desired hypoglycemia, and subtracting from it the amount of exogenous glucose infused. It is now recognized that this approach is not fully quantitative, as underestimates of total R_a and the rate of glucose disposal (R_d) can be obtained. Using a highly purified tracer and taking measurements under steady-state conditions (i.e., constant specific activity) in the presence of low glucose flux eliminates most, if not all, of the problems. In addition, to maintain a constant specific activity, isotope delivery was increased proportionally with increases in exogenous glucose infusion.

Plasma glucose was measured in duplicate by the glucose oxidase technique on a Beckman glucose analyzer. Catecholamines were determined by HPLC (34) with an interassay coefficient of variation (CV) of 12% for both epinephrine and norepinephrine. We made two modifications to the procedure for catecholamine determination: 1) we used a five-point rather than a one-point standard calibration curve, and 2) we spiked the initial and final samples of plasma with known amounts of epinephrine and norepinephrine so that accurate identification of the relevant catecholamine peaks could be made. Corticosterone (ICN Biomedicals, Irvine, CA; interassay CV 7%), insulin interassay (CV 11%) (35), glucagon (Linco Research, St. Louis, MO; interassay CV 15%), and DHEA-S (Diagnostic Systems, Webster, TX; interassay CV 5%) were all measured using radioimmunoassay techniques.

Data are expressed as means ± SE and were analyzed using standard, parametric, one-way ANOVA with repeated measures. A Tukey’s post hoc analysis was used to delineate statistical significance. P ≤ 0.05 was accepted as statistically significant.

Plasma glucose levels were similar during morning and afternoon day-1 euglycemic clamps in the ANTE EUG and ANTE EUG + DHEA-S groups (6.2 ± 0.2 and 6.1 ± 0.2 mmol/l) and during hypoglycemic clamps in the ANTE HYPO and ANTE HYPO + DHEA-S groups (2.8 ± 0.1 and 2.8 ± 0.1 mmol/l). Glucose levels were also similar during day-2 hypoglycemic clamps in all groups (2.8 ± 0.1 mmol/l) (Fig. 2). In addition, day-1 and -2 insulin levels were similar in all four groups of rats (day 1: 726 ± 38 pmol/l; day 2: 750 ± 28 pmol/l for final 30 min).

The increase in plasma norepinephrine during the final 30 min of day-2 hypoglycemia was significantly lower in the ANTE HYPO versus the ANTE HYPO + DHEA-S group (2.0 ± 0.2 vs. 3.3 ± 0.6 nmol/l; F = 7.31; P < 0.05) (Fig. 3). Also, the increase in epinephrine levels during the final 30 min of day-2 hypoglycemia was significantly lower in the ANTE HYPO group versus the ANTE EUG, ANTE EUG + DHEA-S, and ANTE HYPO + DHEA-S groups (8 ± 1 vs. 17 ± 2, 14 ± 3, and 15 ± 3 nmol/l; F = 8.0; P < 0.0001) (Fig. 3). Similarly, glucagon and corticosterone responses during the final 30 min of day-2 hypoglycemia were significantly lower in the ANTE HYPO group versus the ANTE EUG, ANTE EUG + DHEA-S, and ANTE HYPO + DHEA-S groups (91 ± 8 vs. 273 ± 36, 231 ± 42, and 297 ± 78 ng/l; and 1,255 ± 93 vs. 1,915 ± 212, 1,557 ± 112, and 1,668 ± 119 nmol/l, respectively; F = 4.37; P < 0.01) (Fig. 4).

Specific activity was stable during both the basal and the final 30-min periods for all groups, with a CV of <5 ± 1% (Table 1). EGP was significantly lower in the ANTE HYPO group versus the ANTE EUG, ANTE EUG + DHEA-S, and ANTE HYPO DHEA-S groups (13 ± 5 vs. 32 ± 3, 38 ± 7, and 29 ± 8 μmol/l · kg⁻¹ · min⁻¹; F = 7.3; P < 0.001) (Fig. 5). The R_d was similar among groups and did not change during hypoglycemia (Fig. 5). As a consequence of the lower EGP, the exogenous glucose infusion rate needed to maintain the glycemic level during the clamp was significantly greater in the ANTE HYPO group compared with the ANTE EUG, ANTE EUG + DHEA-S, and ANTE HYPO-DHEA-S groups (57 ± 8 vs. 18 ± 3, 18 ± 6, and 22 ± 5 μmol/l · kg⁻¹ · min⁻¹; F = 13.43; P < 0.0001) (Fig. 5).

In the current study, we compared the counterregulatory responses of four groups of rats during 2 h of hyperinsulinemic hypoglycemia on day 2 after different interventions on day 1. The day-1 interventions included antecedent euglycemia, antecedent euglycemia plus DHEA-S infusion, antecedent hypoglycemia, and antecedent hypoglycemia plus DHEA-S infusion. Similar to previous findings from human studies, we found in these conscious, unrestrained rats that two 2-h episodes of hypoglycemia blunted neuroendocrine and metabolic responses to a subsequent episode of hypoglycemia occurring 1 day later. Peripheral infusion of DHEA-S during hypoglycemia on day 1, however, prevented counterregulatory failure and preserved catecholamine, glucagon, corticosterone, and EGP responses to next day hypoglycemia.

In this study, DHEA-S rather than DHEA was infused because the former is water soluble. Using DHEA-S avoids the potentially confounding variable of using a substance (e.g., alcohol or DMSO) that has independent effects on the central nervous system as a vehicle for administering the DHEA-S. Although the structures of DHEA-S and DHEA differ slightly, studies have shown similar physiological effects of DHEA-S and DHEA (20,21) and in humans the hormones are interconverted at a high rate.

The two euglycemic control groups, ANTE EUG + DHEA-S and ANTE EUG, produced similar neuroendocrine and metabolic counterregulatory responses during day-2 hypoglycemia. This indicated that DHEA-S did not have any direct action on modulating counterregulatory responses during day-2 hypoglycemia. However, DHEA-S infused during day-1 hypoglycemia preserved counterregulatory function during day-2 hypoglycemia. The mechanism for this effect is unknown, as a specific DHEA-S receptor has not been identified. It is interesting that many studies have shown that DHEA and its sulfated ester can antagonize corticosteroid actions (22,24,25,36). The role of glucocorticoids in blunting physiological responses to stress are somewhat controversial, with a number of studies both supporting (11,17,19,37) and contradicting (14) this finding (10,13). Several studies, in addition to our own, have shown that corticosteroids can reduce ANS responses to differing forms of stress in a variety of species, such as rats (37), sheep (17), and dogs (38). However, DHEA-S does not antagonize corticosteroid actions by competitively binding to corticosteroid receptors, at least in hepatocytes (39). In addition, chronically administered DHEA has been shown to prevent stress-induced increases in glucocorticoid receptor number (40), which minimizes the biological effects of the elevated corticosteroids. Brain N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid (GABA) receptors are known to have excitatory and inhibitory effects on neuronal norepinephrine release, and thus ANS function, respectively. Although DHEA-S has been shown to have stimulatory effects on NMDA activity (21) and receptor number (41) and inhibitory effects on GABA receptor activity (42), corticosterone has been found to increase the affinity of GABA receptors (43). Therefore, DHEA-S could be stimulatory to the ANS (stimulatory to NMDA and inhibitory to GABA receptors), whereas cortisol may be inhibitory (i.e., may increase activity of GABA receptors). Future studies should be aimed at determining the mechanism for the protective effect of DHEA-S against repeated hypoglycemia in rats.

A number of the effects observed in the present study with antecedent DHEA-S infusion appear to have been mediated via the ANS. Stimulation of the ANS increases plasma catecholamines, plasma glucagon, and, subsequently, EGP. ANS activation also results in reduced glucose uptake during hypoglycemia. Alterations in glucose flux are critical end points in the defense against hypoglycemia. During acute periods of hypoglycemia, increases in EGP provide the primary defense (44). However, during more prolonged hypoglycemia, both reduced glucose uptake and increased EGP become important (44). It is therefore relevant that during the present study DHEA-S infusion prevented the blunting of EGP during subsequent hypoglycemia. Regulation of glucagon responses during hypoglycemia is complex, with data demonstrating that direct α-cell sensing (45), prevailing insulinemia (46), epinephrine, and the ANS (47) may all modulate levels of the hormone during stress. The finding that DHEA-S infusion preserved glucagon responses during our studies may also indicate an action of the compound on preventing ANS dysfunction during repeated episodes of hypoglycemia. The site of a DHEA-S action that protects the ANS cannot be determined from our study. However, DHEA-S readily crosses the blood-brain barrier and peripheral infusions have been found to affect various sites in the brain (20,23,41). We would therefore speculate that DHEA-S can influence ANS responses via direct brain sensing, but future studies will be needed to determine whether this is in fact the case.

DHEA-S also preserved the response of corticosterone during repeated hypoglycemia, thereby suggesting additional non-ANS-mediated effects. Although DHEA-S may antagonize corticosteroid actions, some (48) but not all (49,50) studies have found that DHEA-S can also decrease corticosteroid levels. If this is correct, it suggests that DHEA-S may have the ability to antagonize multiple levels of glucocorticoid negative feedback loops (i.e., on both the ANS and the hypothalamopituitary adrenal axis).

It is interesting that rats do not secrete DHEA-S. This renders the rat a good animal model for testing the hypothesis that DHEA-S could preserve counterregulatory responses during repeated hypoglycemia because any effects of DHEA-S can be isolated. The fact that DHEA-S was infused before and during hypoglycemia on day 1 may be relevant. However, the goal of the present study was to determine if DHEA-S infused during hypoglycemia on day 1 could prevent hypoglycemia-associated autonomic failure 1 day later. The levels of DHEA-S that we infused peripherally reached 0.14 μmol/l, which is ∼5 times the basal levels found in humans. These levels are also similar with those seen in other studies that demonstrated an anticorticosteroid affect on the brain in rats (23) and with levels found with 100 mg of oral DHEA replacement in elderly (51) and young adult subjects (52). Thus, the finding that DHEA-S preserves counterregulatory responses during repeated hypoglycemia in conscious, unrestrained rats could have implications for future human studies.

In conclusion, the present study demonstrated that in the conscious rat, simultaneous DHEA-S administration during antecedent hypoglycemia preserves neuroendocrine (norepinephrine, epinephrine, glucagon, and corticosterone) and metabolic (EGP) responses to next day hypoglycemia. This preservation of counterregulatory function appears to involve the antagonism of mechanisms responsible for causing blunted counterregulatory responses during repeated hypoglycemia rather than the hormone’s directly stimulating neuroendocrine responses per se.

This work was supported by research grants from the Juvenile Diabetes Research Foundation International and the National Institutes of Health (RO1-DK-45369) and a Diabetes Research and Training Grant (5P60-AM-20593).

We thank Eric Allen, Angelina Penaloza, Pam Venson, and Wanda Snead for their expert technical assistance.