Effect of sex on counterregulatory responses to exercise after antecedent hypoglycemia in type 1 diabetes

Am J Physiol Endocrinol Metab 287: E16 E24, 2004. First published March 2, 2004; 10.1152/ajpendo.00480.2002. Effect of sex on counterregulatory responses to exercise after antecedent hypoglycemia in type 1 diabetes Pietro Galassetti, 1 Donna Tate, 1 Ray A. Neill, 1 Sachiko Morrey, 1 David H. Wasserman, 1 and Stephen N. Davis 1,2 1 Departments of Medicine and Molecular Physiology and Biophysics, Vanderbilt University School of Medicine and 2 Nashville Veterans Affairs Medical Center, Nashville, Tennessee 37232-6303 Submitted 28 May 2002; accepted in final form 4 January 2004 Galassetti, Pietro, Donna Tate, Ray A. Neill, Sachiko Morrey, David H. Wasserman, and Stephen N. Davis. Effect of sex on counterregulatory responses to exercise after antecedent hypoglycemia in type 1 diabetes. Am J Physiol Endocrinol Metab 287: E16 E24, 2004. First published March 2, 2004; 10.1152/ajpendo.00480. 2002. A marked sexual dimorphism exists in healthy individuals in the pattern of blunted neuroendocrine and metabolic responses following antecedent stress. It is unknown whether significant sexrelated counterregulatory differences occur during prolonged moderate exercise after antecedent hypoglycemia in type 1 diabetes mellitus (T1DM). Fourteen patients with T1DM (7 women and 7 men) were studied during 90 min of euglycemic exercise at 50% maximal O 2 consumption after two 2-h episodes of previous-day euglycemia (5.0 mmol/l) or hypoglycemia of 2.9 mmol/l. Men and women were matched for age, glycemic control, duration of diabetes, and exercise fitness and had no history or evidence of autonomic neuropathy. Exercise was performed during constant basal intravenous infusion of regular insulin (1 U/h) and a 20% dextrose infusion, as needed to maintain euglycemia. Plasma glucose and insulin levels were equivalent in men and women during all exercise and glucose clamp studies. Antecedent hypoglycemia produced a relatively greater (P 0.05) reduction of glucagon, epinephrine, norepinephrine, growth hormone, and metabolic (glucose kinetics) responses in men compared with women during next-day exercise. After antecedent hypoglycemia, endogenous glucose production (EGP) was significantly reduced in men only, paralleling a reduction in the glucagon-to-insulin ratio and catecholamine responses. In conclusion, a marked sexual dimorphism exists in a wide spectrum of blunted counterregulatory responses to exercise in T1DM after prior hypoglycemia. Key neuroendocrine (glucagon, catecholamines) and metabolic (EGP) homeostatic responses were better preserved during exercise in T1DM women after antecedent hypoglycemia. Preserved counterregulatory responses during exercise in T1DM women may confer greater protection against hypoglycemia than in men with T1DM. sexual dimorphism; glucagon; catecholamines; glucose clamp THE HUMAN BODY REACTS TO PHYSICAL STRESS by eliciting a series of metabolic, neuroendocrine, and autonomic nervous system homeostatic mechanisms, defined as counterregulatory responses. During stresses such as hypoglycemia and exercise, these responses are qualitatively similar and have the common goal to increase glucose availability to restore euglycemia (during hypoglycemia) or to prevent hypoglycemia (during exercise). Counterregulatory failure during physical stress may result in increased susceptibility to hypoglycemia, an issue particularly relevant for patients with type 1 diabetes mellitus (T1DM), in which a high incidence of hypoglycemia is a common complication of therapeutic regimens. Certain aspects of counterregulatory failure may be irreversible (such as the loss of the glucagon responses to hypoglycemia after a few years of T1DM) (14) or may be induced acutely and reversibly (i.e., epinephrine) by an antecedent episode of stress (2). Previous studies from our laboratory have demonstrated that antecedent hypoglycemia and exercise reciprocally blunt subsequent counterregulatory responses. Our recent studies reported that, after antecedent hypoglycemia, counterregulatory responses to next-day exercise were significantly blunted in both healthy subjects (8) and patients with T1DM (13). In a separate study performed in healthy subjects, exercise was used as the antecedent stimulus, and hypoglycemia was performed on the following day. Again, a widespread blunting of neuroendocrine and metabolic responses to second-day hypoglycemia was observed (11). A more recent study from McGregor et al. (19) also reported that antecedent exercise can blunt some (epinephrine, growth hormone) but not all neuroendocrine responses during subsequent hypoglycemia in healthy individuals. In healthy subjects and T1DM, there is a clear sexually dimorphic pattern of neuroendocrine and metabolic responses to differing stimuli such as prolonged hypoglycemia or physical exercise (7, 9). During these conditions, most counterregulatory responses are enhanced in men compared with women (7, 9). Despite this, data from the Diabetes Control and Complications Trial demonstrate that intensively treated women with T1DM have in fact a lowered risk of severe hypoglycemia compared with men (21). Our working hypothesis to answer this apparent clinical paradox is that T1DM women are more resistant to the blunting effects of antecedent hypoglycemia on subsequent counterregulatory responses. Previous studies in healthy humans provide some support for this premise. Episodes of antecedent hypoglycemia or exercise (12) have been demonstrated to blunt counterregulatory responses to subsequent hypoglycemia by a significantly greater extent in healthy men compared with healthy women. The clinical relevance of these findings in healthy subjects, however, has been questioned in a recent editorial (6). This is because, to date, it is unknown whether women with T1DM are also resistant to the blunting effects of antecedent hypoglycemia on counterregulatory responses to subsequent stress. Address for reprint requests and other correspondence: S. N. Davis, 715 PRB, Division of Diabetes, Endocrinology & Metabolism, Vanderbilt Univ. School of Medicine, Nashville, TN 37232-6303 (E-mail: steve. davis@vanderbilt.edu). E16 The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.ajpendo.org

Table 1. Patient characteristics Therefore, to test the hypothesis that antecedent hypoglycemia may result in less blunting of counterregulatory responses during subsequent exercise in T1DM women compared with T1DM men, 14 patients with T1DM (7 female/7 male) were studied during prolonged, moderate exercise after either previous-day euglycemia or two 2-h periods of hypoglycemia of 2.9 mmol/l. RESEARCH DESIGN AND METHODS Males (n 7) Females (n 7) Age, yr 28 3 27 2 Hb A 1C 8.1 0.6 8.0 0.6 BMI, kg/m 2 26 1 24 1 Weight, kg 88 8 69 3* % Body fat 17 2 20 2 Fat mass, kg 15 2 14 1 Fat-free mass, kg 73 0 55 3* V O 2max, ml kg 1 min 1 33 2 28 2 Data are group means SE. BMI, body mass index; V O 2max, maximal O 2 consumption. *P 0.05 vs. males. Subjects. We studied 14 patients (Table 1) with T1DM, seven males and seven females aged 28 2 yr, with a mean body mass index of 25 1 kg/m 2 and glycosylated hemoglobin, or Hb A 1c, of 8.1 0.5% (normal range 4.0 6.5%). Patients had been diagnosed with T1DM 13 2 yr before recruitment and had no evidence of tissue complications of the disease (retinopathy, renal impairment, hypertension) or of diabetic autonomic neuropathy. Each subject had normal blood count, plasma electrolytes, and liver function. All gave written informed consent. Studies were approved by the Vanderbilt University Human Subjects Institutional Review Board. Some of the subjects in this report also participated in a larger study investigating the effects of prior hypoglycemia on subsequent counterregulatory responses during exercise (13). E17 Preliminary testing. At least 2 wk before the initial study, patients body composition was assessed by skinfold caliper technique and whole body plethysmography (Bod-Pod, Life Measurement, Concord, CA). At this time, patients also performed an incremental work test on a stationary cycle ergometer to determine maximal O 2 consumption (V O 2 max) and anaerobic threshold (AT). Air flow and O 2 and CO 2 concentrations in inspired and expired air were measured by a computerized open-circuit indirect calorimetry cart (Medical Graphics Cardio2 cycle) with a mouthpiece and nose-clip system. The AT was determined by the V-slope method (3). The AT determined by gas exchange corresponds to the onset of an increased lactate-to-pyruvate ratio in blood and indicates the level of exercise above which anaerobic mechanisms supplement aerobic energy production (25). At workloads below the AT, exercise can be continued for a prolonged period, whereas above the AT, fatigue will occur considerably faster (24). The experimental work rate was established by calculating 80% AT. The AT was detected at 60 3% of V O 2 max, and 80% AT corresponded to 48 2% of the subjects V O 2 max. 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. Men and women were matched for physical fitness, number of subjects regularly exercising, and baseline levels of prior exercise. The mean V O 2 max was similar for men and women. The mean V O 2 max for the group was 31 2ml kg 1 min 1 (range 21 43 ml kg 1 min 1 ). Experimental design. Each patient was then studied during two separate visits, each lasting two consecutive days and including two overnight stays at our Clinical Research Center (CRC). During day 1 of one of the two visits patients underwent two 2-h morning and afternoon hypoglycemic clamps (Ante Hypo), and during day 1 of the other visit they were maintained euglycemic (Ante Eugly). During day 2 of both visits, all patients performed a similar 90-min exercise protocol under euglycemic conditions. The sequence of Ante Hypo and Ante Eugly studies was randomized, and 6 wk were allowed to elapse between the two visits. Fig. 1. Plasma glucose levels from arterialized venous blood during day 1 [two 120- min clamps with either hypoglycemia of 2.9 mm (Ante Hypo) or euglycemia of 5.0 mm (Ante Eugly)] and glucose and insulin levels from day 2 [90 min of exercise at 50% maximal O 2 consumption (V O 2 max)]. Data are group averages SE; n 14 patients [7 male (M)/7 female (F)] with type 1 diabetes mellitus (T1DM).

E18 Patients were asked to avoid hypoglycemia during the 7 days preceding each visit. Patients checked their blood glucose four times/ day preprandially, before bed, and at any times that they felt a low blood glucose. Plasma glucose values were reported to the investigators so that adjustments of insulin doses and increases in carbohydrate intake could be implemented to avoid hypoglycemia. Detection of any value 70 mg/dl resulted in rescheduling of the study. Patients were also asked to avoid any exercise and consume their usual weightmaintaining diet for 3 days before each study. Each subject was admitted to the Vanderbilt CRC at 4:00 PM on the afternoon before an experiment. Upon admission, patients were asked to discontinue their usual insulin therapy, and two intravenous cannulas were inserted under 1% lidocaine local anesthesia. One cannula was placed in a retrograde fashion into a vein on the back of the left hand. This hand was placed in a heated box (55 60 C) so that arterialized blood could be obtained (23). The other cannula was placed in the contralateral arm so that insulin and 20% glucose (when needed) could be infused via a variable-rate volumetric infusion pump (IMED, San Diego, CA). An insulin infusion was immediately started at a basal rate. Patients then consumed an evening meal and a 7:30 PM snack and were requested not to ingest any food after 10:00 PM. The insulin infusion rate was increased during meal consumption. Through the night, blood glucose was measured every 30 min, and the insulin infusion rate was constantly adjusted to maintain glycemic levels of 4.4 6.6 mmol/l. Day 1 procedures. Day 1 procedures started at 8:00 AM after a 10-h overnight fast and lasted 480 min, divided into an equilibration period (0 120 min), a morning hyperinsulinemic clamp period (120 240 min), a rest period (240 360 min), and an afternoon hyperinsulinemic clamp period (360 480 min). At t 120 min, in all studies a primed continuous infusion of insulin (9 pmol kg 1 min 1 ) was started (20). In prior hypoglycemia studies, plasma glucose was allowed to fall over a 30-min period to a target hypoglycemic plateau of 2.9 mm. Plasma glucose was measured every 5 min and maintained at the desired level via a variable-rate infusion of 20% dextrose (2). In prior euglycemia studies, plasma glucose was held constant at 5.0 mm by a similar technique (10). At t 240 min, the insulin infusion was decreased to morning basal rate; euglycemia was restored in prior hypoglycemia studies and maintained in the prior euglycemia studies. At t 360 min, euglycemic or hypoglycemic clamps identical to those performed in the morning were repeated. At t 480 min, the insulin infusion was decreased to morning basal rate, euglycemia was restored in prior hypoglycemia studies, and all patients were allowed to consume a standardized meal. Afternoon and night procedures were then identical to those of admission night. Day 2 procedures. Day 2 procedures started at 8:00 AM after a 10-h overnight fast and lasted 210 min (t 120 min to 90 min), divided into an equilibration period ( 120 to 30 min), a basal period ( 30 to 0 min), and an exercise period (0 to 90 min). A primed (18 Ci) constant infusion (0.18 Ci/min) of [3-3 H]glucose was started at t 120 min and continued throughout the experiment. Exercise consisted of 90 min of continuous pedaling (at 60 70 rpm) on an upright cycle ergometer (Medical Graphics, Yorba Linda, CA) at 80% of the individual s AT( 50% V O 2 max). Plasma glucose was measured every 5 min and maintained equivalent to baseline levels throughout the study via variable-rate infusion of 20% dextrose. In an attempt to reproduce the drop in insulin levels that physiologically occurs with exercise of this intensity, the basal insulin infusion rate was decreased by 40% after the first 30 min of exercise, provided that the resulting reduced rate was 1 U/h. In cases in which a 40% reduction of the basal rate would have resulted in an insulin infusion rate of 1 U/h, a minimum rate of 1 U/h was maintained. Potassium chloride was also infused (5 mmol/h) during exercise. After completion of the exercise protocol, patients consumed a meal and were discharged. Tracer methodology. Rates of glucose appearance (R a), endogenous glucose production (EGP), and glucose utilization were calculated according to the methods of Wall et al. (22). EGP was calculated by determining the total rate of R a (which comprises both EGP 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 R a and glucose disposal (R d) can be obtained. This underestimate can be largely overcome by use of an HPLC-purified tracer and by measurements taken under steady-state conditions (i.e., constant specific activity). Infusion rates of [3-3 H]glucose were tripled during the first 30 min of exercise to minimize changes in glucose Fig. 2. Incremental (Incr) glucagon (GGN) and catecholamine levels from arterialized venous blood at baseline and during 90 min of exercise at 50% V O 2 max in 14 patients (7M/7F) with T1DM. On the previous day, patients had undergone two 120-min clamps with either hypoglycemia of 2.9 mm (Ante Hypo group) or euglycemia (Ante Eugly group). Data are group averages SE. P 0.05, Ante Hypo vs. Ante Eugly groups as a whole.

E19 specific activity (26). In this study, only data recorded at baseline and during the last 30 min of exercise, when a steady state existed, were used in calculating glucose turnover. Analytical methods. The collection and processing of blood samples have been described elsewhere (5). Plasma glucose concentrations were measured in triplicate by the glucose oxidase method with a glucose analyzer (Beckman, Fullerton, CA). Glucagon was measured according to a modification of the method of Aguilar-Parada et al. (1) with an interassay coefficient of variation (CV) of 12%. Free insulin was measured as previously described (28), with an interassay CV of 9%. Catecholamines were determined by HPLC (4), 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 that accurate identification of the relevant respective catecholamine peaks could be made. Cortisol was assayed using the Clinical Assays Gamma Coat Radioimmunoassay (RIA) kit with an interassay CV of 6%. Growth hormone was determined by RIA (17) with a CV of 8.6%. Pancreatic polypeptide was measured by RIA with the method of Hagopian et al. (15), with an interassay CV of 8%. Lactate, glycerol, alanine, and -hydroxybutyrate were measured in deproteinized whole blood by the method of Lloyd et al. (18). Free fatty acids (FFA) were measured using the WAKO kit adapted for use on a centrifugal analyzer (16). On day 2, blood samples for glucose flux were taken every 10 min throughout the basal period and every 15 min during exercise. Blood for hormones and intermediary metabolites was drawn twice during the basal period and every 15 min during the exercise period. Cardiovascular parameters (pulse and systolic and diastolic arterial pressures) were measured every 10 min from t 30 min to t 90 min. Gas exchange measurements were performed during the basal period and during the final 10 min of exercise. Materials. HPLC-purified [3-3 H]glucose (New England Nuclear, Boston, MA) was used as the glucose tracer (11.5 mci/mm). Human regular insulin was purchased from Eli Lilly (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 means SE, unless otherwise stated, and were analyzed by use of standard, parametric, two-way analysis of variance (ANOVA) with repeated-measures design. This was coupled with Duncan s post hoc test to delineate at which time points statistical significance was reached. A value of P 0.05 indicated significant difference. RESULTS Table 2. Neuroendocrine values at baseline and at end of 90 min of euglycemic exercise at 50% V O 2max in 7 women and 7 men with T1DM Day 1: plasma glucose and insulin levels. Glycemic profiles for the four experimental groups are shown in Fig. 1. Basal plasma glucose levels were comparable in all subjects in both the morning and the afternoon. During hyperinsulinemic clamps, glucose levels remained at baseline in the Ante Eugly subjects and decreased similarly in the Ante Hypo groups (women: morning, 2.9 0.01 mm; afternoon, 2.9 0.01; men: morning, 2.9 0.01; afternoon, 2.9 0.01). Basal plasma insulin levels were similar in all subjects in both the morning (women: Ante Eugly, 60 12 pm; women: Ante Hypo, 60 12 pm; men: Ante Eugly, 55 6 pm; men: Ante Hypo, 60 12 pm) and the afternoon (women: Ante Eugly, 84 18 pm; women: Ante Hypo, 72 18 pm; men: Ante Eugly, 78 24 pm; men: Ante Hypo, 72 18 pm). During hyperinsulinemic clamps, insulin levels increased similarly in all groups of subjects in both the morning (women: Ante Eugly, 570 102 pm; women: Ante Hypo, 540 60 pm; men: Ante Eugly, 624 66 pm; men: Ante Hypo, 546 42 pm) and the afternoon (women: Ante Eugly, 570 114 pm; women: Ante Hypo, 552 42 pm; men: Ante Eugly, 612 60 pm; men: Ante Hypo, 618 72 pm). Day 2: insulin, glucose, and counterregulatory hormone levels. Plasma glucose and insulin (Fig. 1) levels were similar in the four experimental groups before exercise was started, and they remained at basal levels throughout exercise in all subjects. In men, plasma glucagon (Fig. 2, Table 2) increased above basal levels during exercise by 10 2 ng/l after Ante Eugly, whereas after Ante Hypo, glucagon slightly decreased during exercise ( 1 2 ng/l). In women, on the other hand, glucagon increased during exercise by 9 4 ng/l after Ante Eugly and by 4 2 ng/l after Ante Hypo. Therefore, exposure to antecedent hypoglycemia reduced the glucagon response to exercise by 11 2 ng/l in men but only by 5 2 ng/l in women (P 0.05 vs. men). After Ante Eugly, epinephrine increased in men by 639 131 pm during exercise; this increase was reduced to 251 49 pm after Ante Hypo (Fig. 2, Table 2). In women, epinephrine increased by 360 136 pm after Ante Eugly and by 186 60 Baseline End of Exercise Baseline End of exercise Baseline End of exercise Glucagon, ng/l Epinephrine, pm Norepinephrine, nm Women Ante Eugly 40 6 49 7 212 51 572 136 2.0 0.3 4.0 0.7 Ante Hypo 39 6 44 6 225 38 411 78 1.9 0.5 3.9 1.0 Men Ante Eugly 43 2 53 3 256 36 895 112 1.9 0.3 5.2 0.5 Ante Hypo 42 4 41 4 276 36 527 97 1.6 0.3 3.8 0.3 Growth Hormone, g/l Cortisol, nm Pancreatic polypeptide, pm Women Ante Eugly 3 2 13 3 413 113 661 120 14 3 43 14 Ante Hypo 4 2 18 4 492 94 602 130 14 2 36 22 Men Ante Eugly 0.9 0.8 23 10 317 50 620 109 11 1 43 14 Ante Hypo 0.8 0.2 16 5 296 30 462 92 12 2 38 10 Data are means SE. T1DM, type 1 diabetes mellitus; Ante Eugly, euglycemic clamp group; Ante Hypo, hypoglycemic clamp group.

E20 Fig. 3. Incremental pancreatic polypeptide (A), growth hormone (B), and cortisol (C) levels from arterialized venous blood during 90 min of exercise at 50% V O 2 max in 14 patients (7M/7F) with T1DM. On the previous day, patients had undergone two 120-min clamps with either hypoglycemia of 2.9 mm (Ante Hypo group) or euglycemia (Ante Eugly group). Data are group averages SE. P 0.05, Ante Hypo vs. Ante Eugly groups as a whole. Fig. 4. Endogenous glucose production (EGP) and exogenous glucose infusion (GIR) at baseline and during the last 30 min of a 90-min exercise bout at 50% V O 2 max in 14 patients (7M/7F) with T1DM. On the previous day, patients had undergone two 120-min clamps with either hypoglycemia of 2.9 mm (Ante Hypo group) or euglycemia (Ante Eugly group). Data are group averages SE. *P 0.05, Ante Hypo vs. Ante Eugly. pm after Ante Hypo. Ante Hypo therefore blunted the epinephrine response to exercise by 393 49 pm in men but only by 175 60 pm in women (P 0.01). The norepinephrine response to exercise followed a somewhat similar pattern. In men, the exercise-induced increase in norepinephrine was 3.3 0.6 nm after Ante Eugly and 2.2 0.4 nm after Ante Hypo. In women, norepinephrine increased similarly during exercise in both experimental conditions (by 2.0 0.7 nm after Ante Eugly and by 2.0 1.2 nm after Ante Hypo). Ante Hypo therefore induced a significant (P 0.05) blunting of the norepinephrine response to exercise in men but not in women. In men, the exercise-induced increase in growth hormone was 22 11 ng/ml after Ante Eugly and was reduced to 15 6 ng/ml after Ante Hypo (Fig. 3, Table 2). In women, growth hormone increased during exercise by 10 4 ng/ml after Ante Eugly, and this response was moderately increased by Ante Hypo (14 5 ng/ml). The effect of Ante Hypo on the growth hormone response to exercise (a reduction of 7 6 ng/ml in men and an increase of 5 4 g/l in women) was significantly different between sexes (P 0.05). After Ante Eugly, the exercise-induced increase in cortisol was similar in men (303 83 nm) and women (248 83 nm). After Ante Hypo, the cortisol responses were similarly blunted in men (166 110 nm) and women (110 80 nm; Fig. 3). Pancreatic polypeptide responses to exercise were also similar between sexes after Ante Eugly (27 11 pm in women and 22 9 pm in men). The pancreatic polypeptide response to exercise was not affected by Ante Hypo in either sex.

Table 3. Specific activity during basal and final 30-min periods of euglycemic exercise at 50% V O 2max in 7 women and 7 men with T1DM E21 Time (minutes) 30 20 10 0 60 75 90 Male Ante Eugly 385 28 384 23 389 19 363 26 265 23 237 33 242 39 Male Ante Hypo 441 42 435 37 426 39 395 40 254 13 258 18 248 19 Female Ante Eugly 459 61 466 62 441 54 407 47 304 36 282 38 272 32 Female Ante Hypo 471 46 483 63 461 49 414 22 298 38 289 45 264 42 Data are means SE expressed as dpm/ mol. Day 2: glucose kinetics and gas exchange measurements. After day 1 euglycemia, the rate of exogenous glucose infusion (Fig. 4) required to maintain euglycemia during day 2 exercise in men was 9 4 mol kg 1 min 1 and increased to 22 3 mol kg 1 min 1 after Ante Hypo (P 0.05 vs. Ante Eugly). In women, rates of exogenous glucose infusion during the last 30 min were similar after Ante Eugly and after Ante Hypo (12 3 and 16 3 mol kg 1 min 1 ), respectively. Glucose specific activity was stable (CV 4.0%) at the start and at the final 30 min of the day 2 exercise studies (Table 3). Basal rates of EGP were similar in all experimental conditions (Fig. 4). After Ante Eugly, EGP during exercise increased to 29 6 mol kg 1 min 1 in men and to 25 3 mol kg 1 min 1 in women. After Ante Hypo, the EGP response was reduced to 17 9 mol kg 1 m 1 in men (P 0.05 vs. Ante Eugly), whereas it remained unchanged in women (23 6 mol kg 1 min 1 ). Glucose R d during the last 30 min of exercise was not altered in men between experimental conditions (Ante Eugly, 38 4 and Ante Hypo, 39 4 mol kg 1 min 1 ). Glucose R d was similar in women after Ante Hypo (38 7 mol kg 1 min 1 ) and after Ante Eugly (31 5 mol kg 1 min 1 ). Basal rates of RER, lipid oxidation, and carbohydrate oxidation were similar in all experimental groups (Table 4). Rates of lipid and carbohydrate oxidation during exercise in women were unaffected by day 1 hypoglycemia or euglycemia. Conversely, rates of lipid oxidation in men were reduced during the final 15 min of exercise after Ante Hypo (0.9 0.3 Table 4. Gas exchange kinetics and carbohydrate and fat oxidation rates during 90 min of exercise at 50% V O 2max in 7 women and 7 men with T1DM RQ Carbohydrate Oxidation, mg kg 1 min 1 Lipid Oxidation, mg kg 1 min 1 Women, Ante Eugly Baseline 0.85 0.02 1.3 0.3 0.4 0.1 End-exercise 0.93 0.02* 16.3 1.2* 1.3 0.4 Women, Ante Hypo Baseline 0.84 0.02 1.4 0.3 0.6 0.1 End-exercise 0.89 0.02 14.4 1.4* 1.8 0.3 Men, Ante Eugly Baseline 0.88 0.04 2.1 0.5 0.4 0.2 End-exercise 0.94 0.1 17.6 1.1* 1.6 0.1* Men, Ante Hypo Baseline 0.88 0.03 2.0 0.5 0.4 0.1 End-exercise 0.94 0.01* 17.1 0.6* 0.9 0.3 Values are means SE. RQ, respiratory quotient. *P 0.01 vs. corresponding baseline value; P 0.05 vs. Ante Eugly. mg kg 1 min 1 ) compared with Ante Eugly (1.6 0.1 mg kg 1 min 1 ). Day 2: Intermediary metabolism. Blood lactate levels were similar at baseline during all studies (Table 5). The lactate response was significantly reduced by Ante Hypo, and the magnitude of this reduction was similar between sexes. FFA and glycerol basal levels were similar in men and women after both Ante Eugly and Ante Hypo (Table 5). There were no differences in the FFA responses to exercise in men and women after either Ante Eugly or Ante Hypo. No differences in the circulating levels of alanine, glycerol, or the ketone body -hydroxybutyrate were measured between men and women at baseline or during exercise after Ante Eugly or Ante Hypo. Day 2: Cardiovascular parameters. Systolic blood pressure values were higher in men, compared with women, throughout the study (Table 6). Exercise-induced changes in heart rate and systolic, diastolic, and mean arterial pressures, however, were similar between sexes after both Ante Eugly and Ante Hypo. Table 5. Blood lactate, alanine, -HBA, glycerol, and plasma FFA during 90 min of euglycemic exercise at 50% V O 2max in 7 men and 7 women with T1DM Baseline Exercise, min 30 60 90 Blood Lactate Women, Ante Eugly 1.0 0.1 2.9 0.4* 2.5 0.3* 2.5 0.3* Women, Ante Hypo 0.9 0.1 2.0 0.4* 1.8 0.4* 1.6 0.3* Men, Ante Eugly 0.7 0.0 3.4 0.3* 2.4 0.2* 1.9 0.2* Men, Ante Hypo 0.7 0.1 2.6 0.4* 1.8 0.3* 1.5 0.2* Blood Alanine Women, Ante Eugly 0.30 0.03 0.36 0.02 0.39 0.03 0.38 0.03 Women, Ante Hypo 0.35 0.30 0.35 0.03 0.37 0.03 0.36 0.03 Men, Ante Eugly 0.37 0.02 0.39 0.02 0.37 0.02 0.36 0.02 Men, Ante Hypo 0.33 0.02 0.34 0.02 0.34 0.03 0.33 0.02 FFA Women, Ante Eugly 294 46 398 97 419 84 523 122* Women, Ante Hypo 300 42 350 40 371 55 405 63* Men, Ante Eugly 303 35 311 43 379 50 536 76* Men, Ante Hypo 318 64 306 53 324 30 445 27* -HBA Women, Ante Eugly 72 28 53 7 63 8 75 13 Women, Ante Hypo 94 26 51 11 49 11 60 16 Men, Ante Eugly 77 18 51 9 62 15 97 26 Men, Ante Hypo 86 26 58 14 59 10 87 12 Glycerol Women, Ante Eugly 47 7 105 25* 134 32* 171 42* Women, Ante Hypo 57 10 88 12* 111 16* 137 22* Men, Ante Eugly 34 3 74 13* 97 10* 137 9* Men, Ante Hypo 34 4 68 13* 79 14* 109 19* Values are means SE in mm. -HBA, -hydroxybutyrate; FFA, free fatty acids. *P 0.05 vs. corresponding baseline value.

E22 Table 6. Heart rate and arterial systolic, diastolic, and mean blood pressures during 90 min of euglycemic exercise at 50% V O 2max in 7 men and 7 women with T1DM DISCUSSION Baseline Exercise, min 30 60 90 Heart rate, beats/min Women, Ante Eugly 85 8 139 6* 143 7* 149 6* Women, Ante Hypo 83 3 143 4* 146 3* 146 4* Men, Ante Eugly 88 3 146 3* 145 2* 151 4* Men, Ante Hypo 88 5 142 3* 144 2* 146 4* Arterial blood pressure, mmhg Systolic Women, Ante Eugly 111 5 145 7* 144 6* 140 5* Women, Ante Hypo 107 5 136 4* 136 4* 135 4* Men, Ante Eugly # 121 3 155 5* 150 4* 151 4* Men, Ante Hypo # 123 4 152 5 149 5* 150 7* Diastolic Women, Ante Eugly 74 3 70 2 62 3* 65 3* Women, Ante Hypo 71 4 65 2 64 2* 63 1* Men, Ante Eugly 79 3 75 4 71 6* 70 3* Men, Ante Hypo 81 2 76 58 71 5* 70 5* MAP Women, Ante Eugly 84 2 94 3 89 4 90 3 Women, Ante Hypo 83 2 89 2 88 2 87 1 Men, Ante Eugly 94 3 102 3 98 4 97 2 Men, Ante Hypo 95 3 100 5 96 5 95 6 Values are means SE. MAP, mean arterial pressure. *P 0.05 vs. corresponding basal value; men s groups had systolic blood pressure significantly greater (P 0.05) than women s groups. This study was designed to determine whether neuroendocrine and metabolic responses to moderate, prolonged exercise are altered by a sexually dimorphic pattern after antecedent hypoglycemia in patients with T1DM. Our findings show that prior prolonged hypoglycemia (two 2-h bouts, morning and afternoon, at 2.9 mmol/l) reduce glucagon, epinephrine, norepinephrine, growth hormone, and glucose kinetic responses to next day exercise more significantly in T1DM men compared with T1DM women. Experimental conditions were carefully controlled during our 2-day studies. Insulin and glucose levels, relative exercise intensity, and degree of individual physical fitness were equated in men and women. During day 1 and overnight stays, hypoglycemia was carefully avoided (except for the designated hypoglycemic clamp periods) by constant adjustments of exogenous insulin and/or glucose. Additionally, euglycemia was strictly maintained during day 2 exercise. During exercise, hyperglycemia inhibits neuroendocrine responses, whereas hypoglycemia would have induced counterregulatory responses independent of those induced by exercise per se. Furthermore, insulin levels were maintained at basal levels during exercise in all subjects and were identical between sexes. Circulating levels of insulin physiologically decrease during exercise, and the magnitude of this insulin drop may be affected by antecedent stress (8). Preventing insulin differences between experimental groups eliminated an important confounding factor in metabolic (glucose kinetics, fat metabolism) data interpretation. In nondiabetic subjects, the hormonal responses that regulate nutrient flux during submaximal exercise are arranged in hierarchical order, with glucagon, insulin, and epinephrine being the most important. In T1DM, the importance of the glucagon response to exercise is further underscored by the fact that these patients are unable to secrete glucagon in response to insulin-induced hypoglycemia after a disease duration of only a few years (14). In T1DM, secretion of glucagon during exercise is preserved, indicating the stimulus-specific nature of the pancreatic -cell defect. Indeed, in the present study, glucagon increased similarly during exercise in male and female patients after prior euglycemia. After day 1 hypoglycemia, however, the glucagon response to exercise was only moderately attenuated in women, whereas it was completely abolished in men. Epinephrine levels increased similarly in both sexes during exercise after antecedent hypoglycemia. This apparent similarity, however, is in open contrast with the full, nonattenuated response to exercise observed after prior euglycemia, when epinephrine rose significantly more in men than in women. Again, antecedent hypoglycemia exerted a significantly greater blunting effect on epinephrine responses in men than in women. Interestingly, a similar pattern of greater blunting in male patients also occurred with the norepinephrine response to exercise. This parallel reduction in catecholamine responses is consistent with a reduced sympathetic drive in men after prior hypoglycemia, a finding consistent with previous observations following antecedent stress (9, 12). Similar to the response pattern of catecholamine and glucagon to day 2 exercise was the finding that growth hormone responses were more blunted in men compared with women. The cortisol and pancreatic polypeptide responses, on the other hand, were not affected by sex (the cortisol response was similarly blunted in men and women, and the pancreatic polypeptide response was unaffected by antecedent hypoglycemia in either sex). These present results in T1DM are similar to the pattern of neuroendocrine responses occurring after antecedent stress in nondiabetic individuals (5, 10). Pancreatic polypeptide responses reported in this study after antecedent euglycemia (i.e., without the blunting effect of prior hypoglycemia) were not different between sexes and were lower than in nondiabetic subjects exercising under similar experimental conditions (7). Pancreatic polypeptide is considered a marker of vagal efferent input to the pancreas. It has been previously shown that diabetes per se may reduce the pancreatic polypeptide response to stress (hypoglycemia) (27). It is therefore conceivable that prolonged diabetes may also induce a partial inability to appropriately increase pancreatic polypeptide during exercise. In our patients, this may have impaired detection of an effect of sex or of antecedent hypoglycemia on the day 2 pancreatic polypeptide responses. In this present study, differences in neuroendocrine responses after Ante Eugly and Ante Hypo have been presented as absolute values. This convention was used because we believe it is the level of a hormone that elicits a specific physiological response. However, an alternative method of analyzing the data could include computing percentage changes from baseline of the neuroendocrine response during exercise after the differing day 1 challenges. In general, both methods produce similar results. Thus interpretation of the glucagon, norepinephrine, growth hormone, cortisol, and pancreatic polypeptide responses is unaffected by use of either method. The responses of epinephrine and glucagon are more complex and deserve further discussion. In women, epineph-

rine levels increased by 360 pm after Ante Eugly but only 186 pm after Ante Hypo. Thus there was a relative blunting of 48% of epinephrine during exercise after Ante Hypo in T1DM women. In men, epinephrine increased by 640 pm after Ante Eugly but only 360 pm after Ante Hypo. This represented a relative blunting of 61% of epinephrine levels after Ante Hypo in T1DM men. Thus, on one hand, the relative differences in percentage blunting of epinephrine after Ante Hypo in the T1DM men and women was relatively small at 13%. However, the relative reduction in absolute levels of epinephrine in women after Ante Hypo was only 174 pm compared with the much larger 389 pm in T1DM men. Glucagon levels in women increased by 9 ng/l after Ante Eugly but only 5 ng/l after Ante Hypo. This represents a 43% relative blunting of plasma glucagon during exercise after Ante Hypo in women. In the T1DM men, glucagon increased by 10 ng/l after Ante Eugly but did not increase at all after Ante Hypo. This represents a relative blunting of 100% after Ante Hypo compared with Ante Eugly. At first glance, it may appear that the changes in plasma glucagon values in this study are relatively small. However, it should be pointed out that the peripheral plasma values of glucagon during exercise do not accurately reflect the physiologically relevant portal vein levels of the hormone. Thus, due to hepatic extraction, portal vein levels of glucagon during exercise are approximately threefold higher than peripheral values (23). Therefore, the relatively small changes in peripheral values of glucagon would be greatly amplified at the hepatic sinusoid and produce meaningful physiological effects. A significant sexual dimorphism was also present in the metabolic responses to exercise. In T1DM men, the rate of endogenous glucose production during exercise was 11 mol kg 1 min 1 lower following prior hypoglycemia compared with prior euglycemia. This means that prior hypoglycemia obliterated two-thirds of the usual exercise-induced increase in EGP in men, whereas in women this difference was 15% and nonsignificant. As a result, the rates of exogenous glucose infusion required to maintain euglycemia during exercise, which had been similar between sexes after prior euglycemia, became significantly greater in men after hypoglycemia. Consistent with, and a potential mechanism for, the increased glucose infusion rates was the finding that fat oxidation was blunted during exercise after day 1 hypoglycemia in men. On the other hand, fat oxidation during exercise in women was unaffected by day 1 hypoglycemia. Because insulin levels were equivalent during exercise in both groups, the pronounced metabolic changes observed in men after day 1 hypoglycemia are most likely the result of the blunted glucagon and sympathetic nervous system responses. The mechanism(s) responsible for the sexual dimorphism in neuroendocrine responses during exercise after hypoglycemia in T1DM is unknown. Possibilities include inherent differences in men and women (such as sex hormones), differences in body weight, or differential neuroendocrine responses to the antecedent (day 1) hypoglycemia. In summary, two antecedent episodes of hypoglycemia of 2.9 mmol/l induced a clear sexually dimorphic pattern of blunted neuroendocrine and metabolic counterregulatory responses to next-day exercise in patients with T1DM. Glucagon, catecholamines, growth hormone, lipid oxidation, and glucose kinetic responses were more blunted in men than in women. We conclude that women with T1DM are more resistant to the blunting effects of antecedent hypoglycemia on neuroendocrine and metabolic responses to subsequent moderate exercise compared with men. 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