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Sexual Dimorphism in Counterregulatory Responses to Hypoglycemia after Antecedent Exercise

Departments of Medicine and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine and Nashville Veteran Affairs Medical Center, Nashville, Tennessee 37232-6303

Address all correspondence and requests for reprints to: Pietro Galassetti, M.D., Ph.D., 712 MRB II, Division of Diabetes and Endocrinology, Vanderbilt University Medical School, Nashville, Tennessee 37232-6303.

Abstract

After antecedent hypoglycemia, counterregulatory responses to subsequent hypoglycemia exhibit greater blunting in men than in women. Because physical exercise and hypoglycemia share multiple counterregulatory mechanisms, we hypothesized that prior exercise may also result in gender-specific blunting of counterregulatory responses to subsequent hypoglycemia. Thirty healthy subjects (15 women and 15 men; age, 28 ± 3 yr; body mass index, 23 ± 1 kg/m²) were studied during 2-d experiments. Day 1 consisted of either identical 90-min morning and afternoon cycle exercise at 50% maximum oxygen expenditure or two 2-h episodes of hyperinsulinemic euglycemia. Day 2 consisted of a 2-h morning hyperinsulinemic-hypoglycemic clamp. Endogenous glucose production was measured using [3-³H]glucose. Muscle sympathetic nerve activity was measured using microneurography. Day 2 insulin (540 ± 36 pmol/liter) and plasma glucose (2.9 ± 0.06 pmol/liter) levels were similar in men and women during the last 30 min of hypoglycemia. Compared with antecedent euglycemia, d 1 exercise produced significant blunting of d 2 counterregulatory responses to hypoglycemia. Several key d 2 counterregulatory responses were blunted to a greater extent in men than in women: glucagon (men, -105 ± 14; women, -25 ± 7 ng/liter; P < 0.0001), epinephrine (men, -2625 ± 257 pmol/liter; women, -212 ± 573; P < 0.001), norepinephrine (men, -0.50 ± 0.12 nmol/liter; women, -0 ± 0.11; P < 0.001), and muscle sympathetic nerve activity (men, -13 ± 4; women, -4 ± 4 bursts/min; P < 0.01). Cardiovascular responses (heart rate and systolic and mean arterial blood pressures) were also more blunted by antecedent exercise in men than in women. After d 1 exercise, the amount of glucose infused during d 2 hypoglycemia in men was increased 6-fold compared with that after d 1 euglycemia. This amount was significantly increased (P < 0.01) compared with the 2-fold (P < 0.01) increment in glucose infusion that was required in women after d 1 exercise. Lipolysis was unaffected by d 1 exercise in women, but was significantly blunted during d 2 hypoglycemia in men. In summary, two bouts of prolonged, moderate exercise (90 min at 50% maximum oxygen expenditure) induced a marked sexual dimorphism in key neuroendocrine (glucagon, catecholamines, and muscle sympathetic nerve activity) and metabolic (glucose kinetic, lipolysis) responses to next day hypoglycemia.

METABOLIC, NEUROENDOCRINE and autonomic nervous system counterregulatory responses to hypoglycemia and exercise are qualitatively similar. These responses can be reduced by the presence of antecedent stress. Antecedent episodes of hypoglycemia, for instance, can blunt neuroendocrine and metabolic responses to subsequent hypoglycemia (1). The magnitude of this blunting appears to depend on the intensity (1), but not the duration (2), of antecedent hypoglycemia. Quantitatively, the blunting of counterregulatory responses displays a marked sexual dimorphism, with significantly greater blunting occurring in men than in women (3). Similarly, if hypoglycemia precedes next day exercise, counterregulatory responses to exercise are significantly blunted (4), and if the sequence is inverted, i.e. exercise is performed before hypoglycemia (5), counterregulatory responses to second day hypoglycemia are again significantly blunted. It is unknown, however, whether the blunting of counterregulatory responses to hypoglycemia after antecedent exercise also follows a sexually dimorphic pattern. This study was therefore designed to test the hypothesis that antecedent exercise may induce greater blunting of counterregulatory responses to subsequent hypoglycemia in men than in women.

To test this hypothesis, an integrated assessment of counterregulatory responses during hypoglycemia was performed in 30 healthy (15 women and 15 men) subjects after either antecedent rest or two 90-min bouts of submaximal exercise [50% maximum oxygen expenditure (VO₂max)] on a stationary cycle ergometer. On the following morning, subjects underwent a comprehensive assessment of neuroendocrine and metabolic responses to 2 h of hyperinsulinemic hypoglycemia (plasma glucose, 2.8 mmol/liter).

Subjects and Methods

Subjects

Thirty healthy subjects (15 men and 15 women), aged 28 ± 2 yr, with a body mass index of 23 ± 2 kg/m² and glycosylated hemoglobin of 5.5 ± 0.6% (normal range, 4.0–6.5%) volunteered for the study. None was taking any medications or had a family history of diabetes. Each subject had a normal blood count, plasma electrolytes, and liver and renal function, and all gave written informed consent. Studies were approved by the Vanderbilt University human subjects institutional review board. Subjects were asked to consume their usual weight-maintaining diet and to refrain from exercise for the 3 d preceding each study. Female subjects were studied in the midfollicular phase of their menstrual cycles. At 0500 h on the evening before an experiment, subjects were admitted to the Vanderbilt Clinical Research Center. All subjects were studied after an overnight 10-h fast.

Preliminary exercise testing

At least 2 wk before the initial study, subjects performed an incremental work test on a stationary cycle ergometer to determine VO₂max and anaerobic threshold (AT). Air flow and O₂ and CO₂ concentrations in inspired and expired air were measured by a computerized, open circuit, indirect calorimetry cart (Medical Graphics Cardio2, St. Paul, MN) with a mouthpiece and nose clip system. The anaerobic threshold (AT) was determined by the V-slope method (6). The AT determined by gas exchange corresponds to the onset of an increased lactate/pyruvate ratio in blood and indicates the level of exercise above which anaerobic mechanisms supplement aerobic energy production (7). At workloads below the AT, exercise can be continued for a prolonged period, whereas above the AT, fatigue will occur considerably faster (8). Experimental work rate was established by calculating 80% AT. The AT was detected at 59 ± 3% of VO₂max, and 80% AT corresponded to 47 ± 2% of the subject’s VO₂max. This workload was chosen because it is close enough to the AT to produce a physically challenging stress (i.e. large experimental signal), but is sustainable for a prolonged period of time. Subjects studied ranged from sedentary to regularly exercising, although not actively participating in competitive sports. The mean VO₂max for the group was 31 ± 2 ml/kg·min (range, 21–43 ml/kg·min).

Experimental design

Experimental procedures were performed over 2 consecutive days, d 1 and d 2 (Fig. 1). During d 1, 16 subjects (8 men and 8 women, exercise group) performed 2 90-min exercise bouts at about 50%; the remaining 14 subjects (7 men and 7 women, control group) remained at rest for a similar period of time. On d 2 all subjects underwent a 120-min hyperinsulinemic-hypoglycemic clamp (2.8 mmol/liter).

View larger version (23K): [in this window] [in a new window]   Figure 1. Schematic diagram of experimental protocols.

On the morning of d 1, after an overnight fast, two iv cannulas were inserted under 1% lidocaine local anesthesia. One cannula was placed in a retrograde fashion into a vein on the back of the hand. This hand was placed in a heated box (55–60 C) so that arterialized blood could be obtained (9). The other cannula was placed in the contralateral arm so that 20% glucose could be infused via a variable rate volumetric infusion pump (I-med, San Diego, CA).

After insertion of the venous cannulas, a period of 90 min was allowed to elapse followed by a 30-min basal period. Subjects from the exercise group then underwent a 90-min morning exercise period (EXE I), an 180-min resting period, and a second 90-min exercise period (EXE II). EXE I and II consisted each of 90-min continuous submaximal exercise (at 60–70 rpm) on an upright cycle ergometer (Medical Graphics, Yorba Linda, CA) at 80% of the individual’s anaerobic threshold (50% VO₂max).

Plasma glucose was maintained equivalent to baseline levels throughout the study by a glucose clamp technique, according to which glucose levels were measured every 5 min during both exercise periods and every 20 min during the rest period in between exercise. A variable infusion of 20% dextrose was adjusted so that plasma glucose levels were held constant at the desired level (10). Potassium chloride was infused at a rate of 5 mmol/h during EXE I and II. During the first 45 min of the resting period between exercises, a drink containing 1.5 g carbohydrate/kg BW was administered orally to replenish glycogen stores depleted during EXE I. At completion of EXE II, subjects consumed a large meal, bedtime snack, and remained in the Clinical Research Centers.

In subjects from the control group, d 1 procedures followed a similar format to the previously described exercise experiments, except that morning and afternoon hyperinsulinemic-euglycemic clamps were performed in lieu of the exercise bouts. At completion of the second euglycemic clamp subjects consumed an identical evening meal and snack compared with the exercise group.

Day 2 procedures

Day 2 experiments involved a standardized hyperinsulinemic-hypoglycemic glucose clamp to assess the effects of d 1 physical activity on neuroendocrine, autonomic nervous system, and metabolic counterregulatory responses to subsequent hypoglycemia. Day 2 procedures started at 0800 h, after a 10-h overnight fast, and lasted 240 min (-120 to 120 min), divided into an equilibration period (-120 to -30 min), a basal period (-30 to 0 min), and an experimental period (0–120 min). A primed (18 µCi) constant infusion (0.18 µCi/min) of [3-³H]glucose was started at -120 min and continued throughout the experiment. Between -30 and 0 min, basal samples were drawn. At time zero, a primed continuous infusion of insulin (9 pmol/kg·min) was started (11), and the rate of fall of plasma glucose was controlled so that the target hypoglycemic plateau (2.9 mmol/liter) was reached by 30 min and maintained until the end of the experimental period.

Rates of glucose appearance (Ra), endogenous glucose production (EGP), and glucose utilization were calculated according to the methods described by Wall et al. (12). EGP was calculated by determining the total rate of Ra (this comprises both endogenous glucose production and any exogenous glucose infused to maintain euglycemia) 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 Ra and glucose disposal can be obtained. This underestimate can be largely overcome by use of a highly purified tracer and taking measurements under steady state conditions (i.e. constant specific activity), as was done in the present experiments.

Part of the data from the prior rest group were included in related publications from our laboratory (1, 3). Data from individuals in the prior EXE group have been included [not separated by gender, in a previous report (5)].

Direct measurement of muscle sympathetic nerve activity

During d 2 hypoglycemic clamps, muscle sympathetic nerve activity was assessed directly via microneurography. Microneurographic activity was recorded from the peroneal nerve at the level of the fibular head (13). The approximate location of this nerve was determined by transdermal electrical stimulation (10–60 V, 0.01-ms duration). The stimulation produced painless muscle contraction of the foot. After this, a reference tungsten electrode with a shaft diameter of 200 µm was placed sc. A similar electrode with an uninsulated tip (1–5 µm) was inserted into the nerve and used for recording muscle sympathetic nerve activity (MSNA). A recording of MSNA was considered adequate when 1) electrical stimulation produced muscle twitches, but not paresthesia; 2) stretching of the tendons in the foot evoked propiroceptive afferent signals, whereas cutaneous stimulation by slightly stroking of the skin did not; 3) nerve activity increased during phase II of the Valsalva maneuver (hypotensive phase) and was suppressed during phase IV (blood pressure overshoot); and 4) nerve activity increased in response to held expiration.

Two types of sympathetic fibers (skin and muscle) can be identified from recordings of peripheral nerves. MSNA was recorded in the present study, as this has been demonstrated to reflect increased sympathetic activity during insulin-induced hypoglycemia (14), 2-deoxyglucuose-induced neuroglycopenia (15), and hyperinsulinemic euglycemia in normal humans (16).

Sympathetic nerve activity is expressed as bursts per min. Measurements of MSNA were made from the original tracings using a digitizer tablet (HIPAD, Houston Instruments, Austin, TX) coupled to SigmaScan Software (Jandel Scientific, Coite Modena, CA) in a microcomputer. MSNA tracings were analyzed by an observer blinded to the exercise protocol.

Analytical methods

The collection and processing of blood samples were described previously (17). Plasma glucose concentrations were measured in triplicate using the glucose oxidase method with a glucose analyzer (Beckman Coulter, Inc., Fullerton, CA). Glucagon was measured according to a modification of the method of Aguilar-Parada et al. with an interassay coefficient of variation (CV) of 12% (18). Insulin was measured as previously described (19) with an interassay CV of 9%. Catecholamines were determined by HPLC (20) with an interassay CV of 12% for epinephrine and 8% for 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 accurate identification of the relevant respective catecholamine peaks could be made. Cortisol was assayed using the Clinical Assays Gamma Coat RIA kit with an interassay CV of 6%. GH was determined by RIA (21) with a CV of 8.6%. Pancreatic polypeptide was measured by RIA using the method of Hagiopian et al. (22) with an interassay CV of 8%. Lactate, glycerol, alanine, and ß-hydroxybutyrate were measured in deproteinized whole blood using the method of Lloyd et al. (23). Nonesterified fatty acids were measured using the WAKO kit (Richmond, VA) adopted for use on a centrifugal analyzer (9).

Blood samples for glucose flux were taken every 10 min throughout the basal period and every 15 min during the experimental period. Blood for hormone and intermediary metabolite determinations were drawn twice during the basal period and every 15 min during the experimental period. Cardiovascular parameters (pulse and systolic, diastolic, and mean arterial pressures) were measured noninvasively by a Dinamap (Critikon, Tampa, FL) every 10 min from -120 to 120 min. Gas exchange measurements were performed during the basal period and the final 30 min of exercise.

Hypoglycemic symptoms were quantified using a previously validated semiquantitative questionnaire (24). Each individual was asked to rate his/her experience of the symptoms twice during the basal period and every 15 min during the hypoglycemic clamp. Symptoms measured included tiredness, confusion, hunger, dizziness, difficulty thinking, blurry vision, sweating, tremor, agitation, sensation of heat/thirst, and pounding heart. The ratings of the first six symptoms were summed to get a neuroglycopenic score, whereas the rating from the last five symptoms provided an autonomical symptom score.

Materials

HPLC purified [3-³H]glucose (NEN Life Science Products, Boston, MA) was used as the glucose tracer (11.5 mCi/mM). Human regular insulin was purchased from Eli Lilly & Co. (Indianapolis, IN). The insulin infusion solution was prepared with normal saline and contained 3% (vol/vol) of the subject’s own plasma.

Statistical analysis

Data are expressed as the mean ± SE unless otherwise stated and were analyzed using standard, parametric, two-way ANOVA with repeated measures design. This was coupled with Newman-Keuls post-hoc test to delineate at which time points statistical significance was reached. P < 0.05 was considered indicative of a significant difference.

Results

Day 1: plasma glucose and insulin levels

Plasma glucose levels were similarly maintained at basal levels during d 1 exercise in men (5.1 ± 0.11 mmol/liter) and women (5.2 ± 0.11 mmol/liter). These data were comparable to glucose levels measured during d 1 in the control group (men, 5.3 ± 0.17 mmol/liter; women, 5.1± 0.17 mmol/liter). Insulin levels were identical between genders throughout d 1 in the exercise group (men: morning baseline, 42 ± 6 pmol/liter; end of EXE I, 24 ± 6 pmol/liter; afternoon baseline, 48 ±12 pmol/liter; end of EXE II, 30 ± 6 pmol/liter; women: morning baseline, 30 ± 6 pmol/liter; end of EXE I 24 ± 6 pmol/liter; afternoon baseline, 48 ± 12 pmol/liter; end of EXE II, 30 ± 6 pmol/liter). In resting controls insulin levels were as follows: in men: morning baseline, 36 ± 6 pmol/liter; end of morning steady state, 5.2 ± 60 pmol/liter; afternoon baseline, 66 ± 18 pmol/liter; end of afternoon steady state, 540 ± 84 pmol/liter; and in women: morning baseline, 48 ± 12 pmol/liter; end of morning steady state, 426 ± 60 pmol/liter, afternoon baseline, 54 ± 18 pmol/liter; end of afternoon steady state, 744 ± 84 pmol/liter.

Day 2: insulin, glucose, and counterregulatory hormone levels

After d 1 exercise, insulin infusion resulted in equivalent steady state levels by 30 min in both men (546 ± 30 pmol/liter) and women (528 ± 48 pmol/liter; Fig. 2); these levels were comparable to those in the control group (men, 588 ± 30 pmol/liter; women, 546 ± 42 pmol/liter). Plasma glucose fell at an equivalent rate (1.4 mg/min) and reached steady state hypoglycemic plateaus of 3.0 ± 0.056 mmol/liter in men and 2.9 ± 0.056 mmol/liter in women after exercise, and 2.9 ± 0.056 mmol/liter in men and 3.0 ± 0.056 mmol/liter in women after rest (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Figure 2. Plasma glucose and insulin levels from arterialized venous blood during 90-min hypoglycemia (3.0 mmol/liter) in 30 healthy human subjects (15 men and 15 women). On the previous day, 16 subjects (8 men and 8 women, ante-exercise) had undergone two 90-min cycle exercise bouts at about 50% VO2max; the remaining 14 subject (7 men and 7 women, ante-rest) on the previous day were maintained in resting conditions. Data are group averages ± SEM. Data from the ante-exercise groups appeared in combined form in a previous report (5 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Plasma epinephrine, norepinephrine, and glucagon levels from arterialized venous blood during 90-min hypoglycemia (3.0 mmol/liter) in 30 healthy human subjects (15 men and 15 women). On the previous day, 16 subjects (8 men and 8 women, ante-exercise) had undergone two 90-min cycle exercise bouts at about 50% VO2max; the remaining 14 subject (7 men and 7 women, ante-rest) on the previous day were maintained in resting conditions. Data are group averages ± SEM. *, P < 0.05, men vs. women. Data from the ante-exercise groups appeared in combined form in a previous report (5 ).

After exercise, ACTH increased from 8.4 ± 1.1 to 13 ± 1.8 pmol/liter in men and from 9.0 ± 1.1 to 17 ± 2.0 pmol/liter in women (Fig. 4). This was significantly blunted compared with control values for both genders. The extent of blunting, however, was significantly greater in men than in women. Plasma cortisol, on the other hand (Fig. 4), displayed similar increases during d 2 hypoglycemia in both genders after exercise (221 ± 28 to 690 ± 55 nmol/liter in men; 221 ± 55 to 717 ± 83 nmol/liter in women), which was not different from the d 2 cortisol response in control subjects. The GH and pancreatic polypeptide responses were blunted to a similar degree in men and women by antecedent exercise compared with those in the control group (Fig. 4).

View larger version (27K): [in this window] [in a new window]   Figure 4. Plasma pancreatic polypeptide, GH, ACTH, and cortisol levels from arterialized venous blood during 90-min hypoglycemia (3.0 mmol/liter) in 30 healthy human subjects (15 men and 15 women). On the previous day, 16 subjects (8 men and 8 women, ante-exercise) had undergone two 90-min cycle exercise bouts at about 50% VO2max; the remaining 14 subject (7 men and 7 women, ante-rest) on the previous day were maintained in resting conditions. Data are group averages ± SEM. *, P < 0.05, men vs. women. Data from the ante-exercise groups appeared in combined form in a previous report (5 ).

Day 2: MSNA (Fig. 5)

After d 1 exercise basal MSNA was similar in both genders (32 ± 3 bursts/min in men; 31 ± 2 bursts/min in women). During the last 30 min of hypoglycemia MSNA remained unchanged in both genders (men, +2 ± 1 bursts/min; women, +1 ± 1 bursts/min). In the control group hypoglycemia produced an increase in MSNA of 15 ± 5 bursts/min in men and 5 ± 5 bursts/min women. After exercise, therefore, MSNA was significantly more blunted in men than in women (P < 0.05).

View larger version (28K): [in this window] [in a new window]   Figure 5. MSNA during 90-min hypoglycemia (3.0 mol/liter) in 30 healthy human subjects (15 men and 15 women). On the previous day, 16 subjects (8 men and 8 women, ante-exercise) had undergone two 90-min cycle exercise bouts at about 50% VO2max; the remaining 14 subject (7 men and 7 women, ante-rest) on the previous day were maintained in resting conditions. Data are group averages ± SEM. *, P < 0.05, men vs. women. Data from the ante-exercise groups appeared in combined form in a previous report (5 ).

Day 2: glucose kinetics (Fig. 6) and indirect calorimetry

Glucose specific activity (disintegrations per min/mg) was in a steady state during the basal period and the final 30 min of d 2 hypoglycemic clamps. After exercise, the rate of exogenous glucose infusion needed to maintain the target level of hypoglycemia was 0.8 ± 0.3 mg/kg·min in women and 1.3 ± 0.7 mg/kg·min in men. In the control group the glucose infusion rates were 0.4 ± 0.2 mg/kg·min in women and 0.1 ± 0.1 mg/kg·min in men. After d 1 exercise, the rate of glucose disappearance increased during hypoglycemia from 1.9 ± 0.2 to 2.6 ± 0.7 mg/kg·min in men and from 1.6 ± 0.2 and 1.9 ± 0.2 mg/kg·min in women. In the control group the rate of glucose disappearance was unchanged during hypoglycemia in both genders (baseline: men, 1.9 ± 0.2; women, 2.3 ± 0.5 mg/kg·min; last 30 min of hypoglycemia: men, 1.9 ± 0.1; women, 2.3 ± 0.3 mg/kg·min). After d 1 exercise, EGP was similarly suppressed during the last 30 min of hypoglycemia in both genders (from 1.6 ± 0.2 to 1.0 ± 0.2 mg/kg·min in women and from 1.9 ± 0.1 to 1.3 ± 0.2 mg/kg·min in men). In the control group EGP remained similar to baseline in men (from 2.0 ± 0.1 to 1.9 ± 0.1 mg/kg·min), but fell in women from 1.9 ± 0.1 to 1.4 ± 0.2 mg/kg·min).

View larger version (32K): [in this window] [in a new window]   Figure 6. EGP and glucose infusion rate at baseline and during the last 30 min of 2-h hypoglycemia (3.0 mmol/liter) in 30 healthy human subjects (15 men and 15 women). On the previous day, 16 subjects (8 men and 8 women, ante-exercise) had undergone two 90-min cycle exercise bouts at about 50% VO2max; the remaining 14 subject (7 men and 7 women, ante-rest) on the previous day were maintained in resting conditions. Data are group averages ± SEM. *, P < 0.05, men vs. women. Data from the ante-exercise groups appeared in combined form in a previous report (5 ).

Day 2: intermediary metabolism (Table 1)

Blood lactate and alanine levels were similar between genders after d 1 exercise both at baseline and during hypoglycemia. After rest, alanine levels were also similar between genders, whereas lactate increased significantly less in women than in men during hypoglycemia. After d 1 exercise, basal levels of FFA, glycerol, and ß-hydroxybutyrate were higher in women than in men; FFA concentrations and ß-hydroxybutyrate levels decreased to similar levels in both genders during hypoglycemia. The decrease in FFA during hypoglycemia measured after exercise was significantly greater than that observed after rest in both genders. After d 1 exercise, the glycerol response was significantly attenuated in men (from 39 ± 5 to 26 ± 3 mM; P < 0.01), but was maintained at basal levels in women (from 55 ± 6 to 49 ± 11 mM).

Total hypoglycemic symptom scores were similar in men and women after exercise both at baseline (16 ± 2 in both groups) and during the last 30 min of hypoglycemia (34 ± 7 in males and 36 ± 7 in females). These values were similar to those observed after antecedent rest (baseline: men, 16 ± 2; women, 16 ± 1; last 30 min of exercise: men, 40 ± 9; women, 36 ± 9).

Day 2: cardiovascular parameters

After antecedent rest, men displayed greater heart rate, systolic and mean arterial pressure responses to d 2 hypoglycemia compared with women (Table 2). Antecedent exercise appeared to blunt cardiovascular responses in men only, so that gender differences were eliminated (heart rate, mean arterial pressure) or greatly reduced (systolic arterial pressure; Table 2).

The aim of this study was to ascertain whether a sexual dimorphism exists in neuroendocrine and metabolic responses to hypoglycemia after antecedent exercise in healthy humans. Our results demonstrate that prior moderate, prolonged exercise (two 90-min cycling bouts, morning and afternoon, at 50% VO₂max) blunts multiple key neuroendocrine (glucagon, epinephrine, norepinephrine, ACTH, and MSNA), metabolic (glucose kinetics and lipolysis) and cardiovascular (heart rate and systolic and mean blood pressures) responses to next day hypoglycemia by a significantly greater magnitude in men than in women.

During hypoglycemia glucagon and epinephrine are the most important hormones involved in the acute compensatory increase in EGP. In the present study the glucagon response to hypoglycemia after exercise was minimally reduced in women compared with men, whereas epinephrine levels increased similarly in both genders. This apparent similarity in hormonal responses, however, is strikingly different compared with the usual, large physiological sexual dimorphism present in nonattenuated neuroendocrine responses to hypoglycemia. To emphasize this point, after antecedent rest, glucagon responses were greater in men than in women by 113 ± 21 ng/liter, or 219 ± 40%, and epinephrine responses were greater in men than in women by 409 ± 115 ng/liter, or 82 ± 23%. Therefore, when compared with the usual full hypoglycemic response, the glucagon responses after exercise were reduced by more than 100 ng/liter in men, but only by about 25 ng/liter in women. Similarly, the epinephrine response to hypoglycemia was reduced by more than 2456 pmol/liter in men and by only 164 pmol/liter in women. Norepinephrine, MSNA, and cardiovascular responses after exercise were also significantly more attenuated in males than in females compared with the usual full counterregulatory responses. It would therefore appear that antecedent exercise can overcome the greater sympathetic nervous system responses to hypoglycemia usually present in men.

During d 2 hypoglycemia, the GH and pancreatic polypeptide responses after exercise were blunted to a similar degree in men and women. Therefore, prior exercise appears to exert selective gender-specific blunting effects on counterregulatory responses to subsequent hypoglycemia. Indeed, the cortisol response to d 2 hypoglycemia, unlike that of other major counterregulatory hormones, was not blunted by antecedent exercise in either gender. ACTH levels, on the other hand, were blunted to a significantly greater extent in men than in women. The dissociation between ACTH and cortisol responses has been previously noted (5). We speculate that the lack of blunting of the cortisol responses is probably not modulated by the anterior pituitary, but by some mechanisms downstream of this gland. Changes in both adrenal sensitivity to ACTH (25) and ACTH sensitivity to glucocorticoids (26) have been reported and may conceptually explain alterations in cortisol levels independent of ACTH release.

Despite the greater blunting of neuroendocrine responses after antecedent exercise in men, hypoglycemic symptom scores were not different during hypoglycemia between genders after rest or exercise. A potential explanation for this observation is that differing antecedent stresses may blunt counterregulatory responses to subsequent hypoglycemia in a hierarchical fashion. This hypothesis is supported by the previous observation that when neuroendocrine responses to subsequent hypoglycemia were blunted by antecedent hypoglycemia, cardiovascular responses were unaffected (1). Further, after very short (5-min) antecedent hypoglycemia (2), metabolic, neuroendocrine, and MSNA responses to subsequent hypoglycemia were blunted, but symptom scores, similar to those in the present study, were not decreased compared with antecedent euglycemia. Additionally, Paramore et al. (27) observed that the forearm sympathetic neural response to subsequent hypoglycemia was preserved after antecedent hypoglycemia, whereas most other neuroendocrine and metabolic responses were attenuated.

Metabolic counterregulatory responses also demonstrated sexual dimorphism. After rest, lactate levels increased significantly less in women than in men. After antecedent exercise, however, lactate levels increased similarly in both genders during d 2 hypoglycemia. The reason for this discrepancy is probably linked to the epinephrine response. After d 1 rest, the epinephrine response in women was about 50% lower than that in men. Epinephrine is the main determinant of gluconeogenic precursor release from peripheral tissues; not surprisingly, therefore, reduced epinephrine responses were paralleled by reduced lactate levels. After exercise, on the other hand, similar epinephrine responses probably determined that lactate levels were also comparable between genders. Lipolytic responses (best quantified by glycerol levels) were significantly blunted by antecedent exercise in men, but were unaffected in women. Lipolysis is an important counterregulatory mechanism that has been demonstrated to contribute about 25% of the body’s total defense against prolonged hypoglycemia (28). The magnitude of the lipolytic response exerts an effect on counterregulation by providing FFAs that inhibit glucose uptake in muscle, and glycerol and FFAs that provide substrate and energy, respectively, for hepatic gluconeogenesis. Thus, the size of the lipolytic response can help modulate the amount of glucose that is required to preserve glucose levels during a hypoglycemic clamp. In these present studies the amount of glucose that was required to maintain the required hypoglycemia was dramatically increased in men (6-fold) after antecedent exercise. This compared with only a 2-fold increase in glucose infusion rates in women. Therefore, the greater blunting of neuroendocrine and sympathetic nervous system counterregulatory responses in men was translated into a significant metabolic deficit that required a substantially increased amount of glucose to protect the men during hypoglycemia.

Our observations may have relevance for patients with type I diabetes. The reduction in long-term tissue complications (29) that was achieved in these patients through strict glycemic control came at the cost of an increased prevalence of hypoglycemia (29). Imperfect insulin replacement and permanent loss of the glucagon response to hypoglycemia (30) are probably partly responsible for this complication of intensive diabetic treatment. It is now believed, however, that antecedent hypoglycemia also acts as an additional acquired form of counterregulatory failure (31, 32, 33, 34). In nondiabetic subjects we have previously demonstrated that after antecedent hypoglycemia, blunting of counterregulatory responses to subsequent hypoglycemia is significantly more pronounced in males than in females. With the present study we showed that this is also true when hypoglycemia follows antecedent exercise. Future studies are needed to confirm whether this sexual dimorphism also occurs in diabetic subjects.

The pattern of gender differences reported in the present study is similar to the sexual dimorphism that we have previously observed when counterregulatory responses to hypoglycemia were measured after antecedent hypoglycemia (1), suggesting that common mechanisms may be at work in both conditions. Direct comparison of the present data with work from other laboratories, on the other hand, is difficult. The only other study that, to our knowledge, has investigated the effects of exercise on subsequent hypoglycemia studied a relatively small number of subjects (three women and four men), and the data were reported as a group without reference to gender responses (35).

In summary, two antecedent bouts of prior prolonged, moderate cycling exercise, resulted in blunting of key neuroendocrine and metabolic (glucagon, catecholamines, MSNA, glucose kinetics, and lipolysis) responses to next day hypoglycemia that was significantly greater in males than in females.

We conclude that in overnight fasted healthy humans, antecedent exercise can alter neuroendocrine and metabolic counterregulatory responses to subsequent hypoglycemia in a clear gender-specific fashion. Neuroendocrine and metabolic counterregulatory responses in women are significantly less blunted after antecedent exercise compared with those in men. This sexual dimorphism in counterregulatory responses is similar to that observed after antecedent hypoglycemia.

Acknowledgments

We thank Eric Allen, Angelina Penalosa, and Wanda Snead for expert technical assistance. We also appreciate the skill and help of the nurses of Vanderbilt General Clinical Research Center in the performance of the studies included in this report.

Footnotes

This work was supported by a grant from the Juvenile Diabetes Foundation International, NIH Grant R01-DK-45369, Diabetes Research and Training Center Grant 5P60-AM-20593, Clinical Research Center Grant M01-RR-00095, and a V.A./Juvenile Diabetes Foundation Institute Diabetes Research Center grant.

Abbreviations: AT, Anaerobic threshold; CV, coefficient of variation; EGP, endogenous glucose production; EXE, exercise period; MSNA, muscle sympathetic nerve activity; Ra, rate of glucose appearance; VO₂max, maximum oxygen expenditure.

Received January 9, 2001.

Accepted April 17, 2001.

References

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