THE CENTRAL IMPORTANCE of prior hypoglycemia in

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1 X/01/$03.00/0 Vol. 86, No. 5 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2001 by The Endocrine Society Effects of Morning Hypoglycemia on Neuroendocrine and Metabolic Responses to Subsequent Afternoon Hypoglycemia in Normal Man* S. N. DAVIS AND D. TATE Departments of Medicine, Molecular Physiology, and Biophysics, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, Tennessee Received January 19, Revision received January 8, Accepted January 12, Address all correspondence and requests for reprints to: Stephen N. Davis, M.D., Division of Diabetes and Endocrinology, Vanderbilt University School of Medicine, 712 Medical Research Building II, Nashville, Tennessee steve.davis@mcmail.vanderbilt.edu. * This work is supported by a grant from the Juvenile Diabetes Foundation International, NIH Grant R01 DK45369, Diabetes Research and Training Center Grant 5P60-AM20593, Clinical Research Center Grant M01-RR00095, and a VA/JDFI Diabetes Research Center grant. ABSTRACT There is general agreement that prior hypoglycemia blunts subsequent hypoglycemic counterregulatory responses. However, there is considerable debate concerning the timing and number of prior hypoglycemic episodes required to cause this blunting effect. The aim of this study was to determine whether one episode of hypoglycemia could modify neuroendocrine, metabolic, and symptom responses to hypoglycemia induced 2 h later. A total of 24 (12 male and 12 female) young, healthy, overnight-fasted subjects participated in a series of glucose clamp studies. A total of 16 individuals underwent 2 randomized studies of either identical 2-h morning and afternoon hyperinsulinemic ( pmol/l) hypoglycemia ( mmol/l) separated by 2 h or, at least 2 months later, 2-h morning and afternoon hyperinsulinemic ( pmol/l) euglycemia ( mmol/l). A total of 8 other subjects participated in a single experiment that consisted of 2-h morning hyperinsulinemic ( pmol/l) euglycemia ( mmol/l) and 2-h afternoon hyperinsulinemic ( pmol/l) hypoglycemia ( mmol/l) also separated by 2 h. Morning hypoglycemia significantly (P 0.01) reduced (33 55%) the responses of epinephrine, norepinephrine, glucagon, GH, cortisol, and pancreatic polypeptide during afternoon hypoglycemia. Hypoglycemic symptoms (primarily neuroglycopenic) were also significantly (P 0.01) reduced during afternoon hypoglycemia. Plasma glucose, insulin, nonesterified fatty acids, glycerol, lactate, -hydroxybutyrate (P 0.01), GH, and cortisol (P 0.05) levels were significantly increased at the start of afternoon hypoglycemia following morning hypoglycemia. Morning hypoglycemia created an insulin-resistant state during afternoon hypoglycemia. Despite blunted neuroendocrine responses, glucose infusion rates required to maintain hypoglycemia and increases in glucose oxidation were significantly attenuated during afternoon compared with morning hypoglycemia. This was in marked contrast to euglycemic control experiments where glucose infusion rates and nonoxidative glucose disposal were significantly increased during afternoon relative to morning studies. We conclude that in normal man one episode of prolonged, moderate, morning hypoglycemia can produce substantial blunting of neuroendocrine and symptomatic responses to subsequent near-term hypoglycemia, and the induction of posthypoglycemic insulin resistance can compensate for blunted neuroendocrine responses by limiting glucose flux and specifically glucose oxidation during subsequent near-term hypoglycemia. (J Clin Endocrinol Metab 86: , 2001) THE CENTRAL IMPORTANCE of prior hypoglycemia in the pathogenesis of hypoglycemic counterregulatory failure is now accepted (1 6). However, there is controversy surrounding the time interval between and number of antecedent hypoglycemic episodes required to produce blunted counterregulatory responses (3 6). Heller and Cryer (4) demonstrated that one episode of hypoglycemia was unable to diminish counterregulatory responses to hypoglycemia induced 24 h later. However, two episodes of same-day hypoglycemia substantially blunted counterregulatory responses to following morning hypoglycemia (i.e. 18 h later) (4). In contrast, Veneman et al. (5) reported that one episode of nocturnal hypoglycemia could significantly reduce all major neuroendocrine responses to subsequent hypoglycemia 8 h later. Davis et al. (3, 6) induced two episodes of hypoglycemia over a 330-min period of continuous hyperinsulinemia in groups of normal individuals and patients with type 1 diabetes. Glucagon (in normals), GH, and cortisol, but not epinephrine or norepinephrine, were blunted during the second hypoglycemic period. The preservation of epinephrine responses during this experimental model was noteworthy as patients with type 1 diabetes are reliant on this catecholamine for adequate counterregulation during hypoglycemia (7). Thus, there is conflicting information regarding whether prior hypoglycemia can blunt any or all counterregulatory responses to a second episode of hypoglycemia occurring within the same day. This has clinical relevance. Intensively treated patients with type 1 diabetes often suffer hypoglycemia before lunch and before supper. Whether the effects of late morning hypoglycemia can feed forward to reduce neuroendocrine responses to late afternoon hypoglycemia is not established. The acute physiologic effects of an episode of prior hypoglycemia on metabolism during subsequent same-day hypoglycemia in normal man have not been determined. The metabolic actions of catecholamines, cortisol, and GH are prolonged and produce a milieu of insulin resistance (8 10). Therefore, although levels of neuroendocrine hormones are important determinants of the counterregulatory response, it is their metabolic end points that maintain homeostasis. Thus, two additional unanswered questions need to be ad- 2043

2 2044 DAVIS AND TATE JCE&M 2001 Vol. 86 No. 5 dressed: 1) to what extent is metabolism altered by prior same-day hypoglycemia, and 2) will an altered metabolic environment modulate actions of counterregulatory hormones during a subsequent, near-term episode of hypoglycemia? Therefore, the aims of this study were to determine whether 2 h of moderate hypoglycemia (50 mg/dl) could diminish neuroendocrine and symptomatic responses to subsequent hypoglycemia induced 2 h later, and to determine how same-day prior hypoglycemia alters intermediary metabolism during subsequent near-term hypoglycemia in normal man. Subjects Materials and Methods We studied 24 healthy volunteers (12 male and 12 female) aged 29 2 yr with a body mass index of 22 1 kg/m 2 and glycosylated hemoglobin (hemoglobin A 1c )of % (normal range, 4 6.5%). None were taking medication or had a family history of diabetes. Each subject had a normal blood count, plasma electrolytes, and liver and renal function. All gave written informed consent. Studies were approved by the Vanderbilt University human subjects Institutional Review Board. The subjects were asked to avoid any exercise and consume their usual weight-maintaining diet for 3 days before each study. Each subject was admitted to the Vanderbilt Clinical Research Center at 1700 h on the evening before an experiment. All subjects were studied after an overnight 10-h fast. Glucose clamp studies A total of 16 subjects attended for two separate randomized experiments (repeat hypoglycemia and repeat euglycemia studies) separated by at least 2 months. Subjects were blinded as to the order of the two glucose clamp studies. A total of 8 additional subjects participated in a single 1-day study (morning euglycemia, afternoon hypoglycemia). On the morning of each study, after an overnight fast, two iv cannulae 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 (11). The other cannula was placed in the contralateral arm so that 20% glucose could be infused via a variable rate volumetric infusion pump (Imed, San Diego, CA). Repeat hypoglycemia experiments After insertion of venous cannulae, a period of 90 min was allowed to elapse followed by a 30-min basal control period and a 120-min hyperinsulinemic-hypoglycemic experimental period. At time zero, a primed continuous infusion of insulin (12) was administered at a rate of 9 pmol/kg min for 120 min. Plasma glucose levels were measured every 5 min, and a variable infusion of 20% dextrose was adjusted so that plasma glucose levels were held constant at mmol/l (13). Potassium chloride was infused at a rate of 5 mmol/h during each study. After completion of the initial 2-h test period, the insulin infusion was discontinued and plasma glucose was rapidly restored to euglycemia with 20% dextrose. Plasma glucose was maintained at euglycemia for a further 2 h by initially adjusting an exogenous glucose infusion. However, by 100 min, the exogenous glucose infusion was discontinued as subjects could maintain euglycemia without external glucose support. Two hours after cessation of the morning experiment, insulin was restarted and a second hyperinsulinemic-hypoglycemic clamp, identical with the morning clamp, was performed. Repeat euglycemia experiments These experiments followed a similar format to the previously described hypoglycemia experiments, with the exception that identical morning and afternoon hyperinsulinemic (9 pmol/kg min) euglycemic (5.0 mmol/l) clamps were performed (14). An exogenous glucose infusion was required to maintain euglycemia during the entire 2-h interval between morning and afternoon glucose clamp studies. Morning euglycemia, afternoon hypoglycemia experiments These experiments also followed a similar format to the above experiments, with the exception that the morning study consisted of a 2-h hyperinsulinemic (9 pmol/kg min) euglycemic (5.0 mmol/l) clamp, followed 2 h later by an afternoon 2-h hyperinsulinemic (9 pmol/ kg min) hypoglycemic (2.9 mmol/l) clamp. As described above, an exogenous glucose infusion was used to maintain euglycemia during the 2-h interval between morning and afternoon glucose clamp studies. Indirect calorimetry Air flow, O 2, and CO 2 concentrations in expired gases were measured by a computerized open-circuit system (Medical Graphics Corp., Yorba Linda, CA). Rates of fat and carbohydrate oxidation and energy expenditure before and during the final 30 min of glucose clamp studies were calculated from rates of measured VO 2 and VCO 2 as described by Frayn (15). Rates of nonoxidative glucose disposal were calculated by subtracting the amount of glucose disappearance as measured by indirect calorimetry from the total glucose infusion rate required to maintain euglycemia (16). Analytical methods The collection and processing of blood samples have been described elsewhere (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. (18) with an interassay coefficient of variation (CV) of 12%. Insulin was measured as previously described (19) with an interassay CV of 9%. Catecholamines were determined by high-performance liquid chromatography (20) with an interassay 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 Hagopian et al. (22) with an interassay CV of 8%. Lactate, glycerol, alanine, and -hydroxybutyrate were measured in deproteinized whole blood using the method of Lloyd et al. (23). Nonesterified fatty acids (FFA) were measured using the WAKO kit (Wako Pure Chemical Industries Ltd., Richmond, VA) adopted for use on a centrifugal analyzer (24). Blood for hormones and intermediary metabolites was drawn twice during the control period and every 15 min during the experimental period. Cardiovascular parameters (pulse, systolic, diastolic, and mean arterial pressure) were measured noninvasively by a Dinamap (Critikon, Tampa, FL) every 10 min throughout each 210-min study. Gas exchange measurements were performed during the control period and during the final 30 min of each glucose clamp. Hypoglycemic symptoms were quantified using a previously validated semiquantitative questionnaire (25). Each individual was asked to rate his/her experience of the symptoms twice during the control period and every 15 min during experimental periods. Symptoms measured included tiredness, confusion, hunger, dizziness, difficulty thinking, blurred vision, sweaty, tremor, agitation, hot/thirsty, and pounding heart. The ratings of the first six symptoms were summed to get a neuroglycopenic score whereas the ratings from the last five symptoms provide an autonomic symptom score. Materials 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 subjects own plasma.

3 NEUROENDOCRINE/METABOLIC RESPONSE TO HYPOGLYCEMIA 2045 Statistical analysis Data are expressed as mean se unless otherwise stated, and analyzed using standard, parametric, two-way ANOVA with repeated measures design. This was coupled with Student s t test to delineate at which time statistical significance was reached. A value of less than 0.05 indicated significant difference. Results Insulin and glucose levels during morning and afternoon hypoglycemia studies Insulin levels increased from 36 6to pmol/l by the final 30 min of hypoglycemia in the morning and by similar amounts (48 12 to pmol/l) during afternoon hypoglycemia. Insulin levels were significantly increased (P 0.05) at the start of afternoon hypoglycemia following morning hypoglycemia (Fig. 1). Plasma glucose levels were also significantly increased at the start of hypoglycemia following morning hypoglycemia ( vs mmol/l; P 0.01). Plasma glucose levels were maintained at mmol/l during morning euglycemia and at mmol/l during all morning and afternoon hypoglycemia studies. Neuroendocrine responses to morning and afternoon hypoglycemia Epinephrine levels increased from basal values of to nmol/l by the final 30 min of morning hypoglycemia. Despite equivalent hypoglycemia, epinephrine levels during the afternoon following morning hypoglycemia ( nmol/l) were significantly blunted (P 0.01) compared with morning values (Fig. 2). Morning euglycemia had no effect on epinephrine responses during afternoon hypoglycemia to nmol/l). Plasma norepinephrine levels (Fig. 2) increased significantly during morning hypoglycemia ( to nmol/l; P 0.01) and during afternoon hypoglycemia following morning euglycemia ( to nmol/l; P 0.05). Following morning hypoglycemia, norepinephrine levels remained similar to baseline ( to nmol/l) during afternoon hypoglycemia. Pancreatic polypeptide responses (Fig. 2) were also significantly greater (P 0.01) during morning (20 3to pmol/l) compared with afternoon hypoglycemia (27 6to88 14 pmol/l). Morning euglycemia had no effect on pancreatic polypeptide responses during afternoon hypoglycemia (18 4 to pmol/l). Plasma glucagon levels in the morning increased from basal values of 58 4to133 8 ng/l (Fig. 3). Morning hypoglycemia resulted in significantly blunted (P 0.01) glucagon responses during afternoon hypoglycemia (51 4 to 73 7 ng/l). Plasma cortisol levels increased from to nmol/l in the morning and from to nmol/l during afternoon hypoglycemia following morning euglycemia. These values were significantly greater (P 0.05) than the response in the afternoon following morning hypoglycemia ( to nmol/l). GH levels FIG. 1. Arterialized insulin and glucose levels during morning and afternoon hyperinsulinemic-hypoglycemic clamps in overnight-fasted normal humans.

4 2046 DAVIS AND TATE JCE&M 2001 Vol. 86 No. 5 FIG. 2. Effects of peripherally infused insulin (9 pmol/kg min) and hypoglycemia ( mmol/l) on arterialized plasma epinephrine, norepinephrine, and pancreatic polypeptide levels during morning and afternoon hypoglycemic glucose clamp studies., Afternoon hypoglycemia epinephrine and pancreatic polypeptide levels are significantly reduced (P 0.01) as compared with morning hypoglycemia and afternoon hypoglycemia following euglycemia values., Norepinephrine repsonses are reduced (P 0.05) during afternoon hypoglycemia as compared with morning hypoglycemia and afternoon hypoglycemia following morning euglycemia. (Fig. 3) increased from 3 1to40 4 g/l during morning hypoglycemia and from 2 1to36 3 g/l during afternoon hypoglycemia following morning euglycemia, which were also significantly greater (P 0.05) than the response following morning hypoglycemia (6 2to32 4 g/l). Basal levels of epinephrine, norepinephrine, glucagon, and pancreatic polypeptide were similar at the start of morning and afternoon hypoglycemia. Cortisol and GH levels were increased (P 0.05) at the start of afternoon compared with morning hypoglycemia. Thus, 2 h of interval euglycemia was sufficient for the majority of counterregulatory hormones to return to usual postabsorptive values. Metabolic responses during hypoglycemia Intermediary metabolism at the start of afternoon experiments was significantly affected by morning hypoglycemia. FIG. 3. Effects of peripherally infused insulin (9 pmol/kg min) and hypoglycemia ( mmol/l) on arterialized plasma glucagon, cortisol, and GH levels during morning and afternoon hypoglycemic glucose clamp studies. Afternoon glucagon (, P 0.01), cortisol (, P 0.05), and GH (, P 0.05) responses are significantly reduced as compared with morning hypoglycemia and afternoon hypoglycemia following morning euglycemia. Despite the significantly elevated insulin and glucose values, plasma FFA ( vs mol/l), blood glycerol (95 10 vs mol/l), and -hydroxybutyrate levels ( vs mol/l) were significantly increased (P 0.05) at the start of afternoon following morning hypoglycemia, respectively. Similarly, blood lactate was also significantly increased at the start of afternoon hypoglycemia compared with morning studies ( vs mol/l; P 0.01). Steady state values of intermediary metabolites were similar during the final 30 min of morning and afternoon hypoglycemia. However, following morning hypoglycemia incremental from baseline metabolic responses during afternoon hypoglycemia were significantly reduced. Thus, there were greater reductions (P 0.05) of FFA ( vs mol/l), glycerol (-32 4 vs. 8 3 mol/ L), and -hydroxybutyrate ( vs mol/l) during afternoon hypoglycemia following morning hypoglycemia. Similarly, the increase in blood lactate was attenuated in the afternoon following morning hypoglycemia

5 NEUROENDOCRINE/METABOLIC RESPONSE TO HYPOGLYCEMIA 2047 TABLE 1. Effects of hyperinsulinemic (9 pmol/kg min) hypoglycemia ( mmol/l) and euglycemia ( mmol/l) on intermediary metabolism in overnight-fasted normal individuals Morning studies duration (min) Afternoon studies duration (min) Blood lactate ( mol/l) Euglycemia Euglycemia Euglycemia Hypoglycemia a a a Hypoglycemia a a Hypoglycemia a,b a a Blood glycerol ( mol/l) Euglycemia Euglycemia Euglycemia Hypoglycemia a 31 2 a 33 3 a Hypoglycemia a 55 5 a 58 7 a Hypoglycemia a,b a,b a,b a,b Blood -hydroxybutyrate ( mol/l) Euglycemia Euglycemia Euglycemia Hypoglycemia Hypoglycemia Hypoglycemia a,b a,b a,b a,b Plasma FFA ( mol/l) Euglycemia Euglycemia Euglycemia Hypoglycemia Hypoglycemia a a a Hypoglycemia a,b a,b a,b a,b Values are mean SEM. a Hypoglycemia values are significantly increased (P 0.01) as compared with euglycemia. b Morning/afternoon hypoglycemia levels are significantly increased (P 0.01) as compared with morning euglycemia/afternoon hypoglycemia. ( vs mol/l; P 0.05). Following morning euglycemia, blood lactate responses during hypoglycemia were preserved but glycerol, -hydroxybutyrate, and FFA levels were attenuated compared with morning hypoglycemia values (Table 1). Despite reduced neuroendocrine responses, glucose infusion rates required to maintain hypoglycemia were lower (P 0.05) during the final 30 min of afternoon hypoglycemia following morning hypoglycemia ( vs mol/kg min), respectively (Table 2). Following morning euglycemia, and despite preserved neuroendocrine responses, glucose infusion rates were significantly increased (P 0.01) during afternoon hypoglycemia ( mol/kg min). Indirect calorimetry during hypoglycemia Respiratory quotient (RQ) was significantly increased at the start of afternoon hypoglycemia following morning hypoglycemia ( vs ; P 0.05). RQ increased significantly during morning hypoglycemia ( to ; P 0.01) but remained similar to baseline during subsequent afternoon hypoglycemia ( to ). Consistent with the changes in RQ, glucose oxidation increased by a greater amount (P 0.01) during morning hypoglycemia ( to mol/kg min) compared with subsequent afternoon hypoglycemia ( to mol/kg min). Lipid disappearance declined to similar levels during morning and afternoon hypoglycemia. Following morning euglycemia, RQ was significantly increased at the start of afternoon hypoglycemia ( vs ; P 0.05). RQ increased significantly during afternoon hypoglycemia following morning euglycemia ( vs ; P 0.05). Consistent with changes in RQ, glucose oxidation increased significantly during afternoon hypoglycemia following morning euglycemia ( to mol/kg min; P 0.05). Hypoglycemic symptom scores Symptom scores were similar at the start of morning and afternoon studies (16 1 vs. 17 2, respectively). Incremental hypoglycemic symptom scores were significantly reduced during afternoon hypoglycemia compared with morning hypoglycemia and euglycemia( 10 1 vs. 24 4, respectively; P 0.01). Increases in hypoglycemic symptom scores ( 20 3) were unaffected by morning euglycemia. Although increases in autonomic symptom scores were reduced during afternoon compared with morning hypoglycemia ( 7 1 vs. 12 2; P 0.05), the greatest blunting was observed in neuroglycopenic symptoms ( 3 1 vs. 12 2; P 0.01). Cardiovascular responses during hypoglycemia Heart rate and systolic and diastolic blood pressure were similar at the start of morning and afternoon hypoglycemia studies. Heart rate increased similarly during morning (59 4to65 4 beats/min) and afternoon studies (59 4to70 5 beats/min). Systolic blood pressure increased similarly during morning (108 4to113 3 mm Hg) and afternoon (105 5to109 5 mm Hg) and diastolic blood pressure

6 2048 DAVIS AND TATE JCE&M 2001 Vol. 86 No. 5 TABLE 2. Glucose infusion rates ( mol/kg min) used to maintain euglycemia ( mmol/l) or hypoglycemia ( mmol/l) during hyperinsulinemia (9 pmol/kg min) in overnight-fasted normal individuals Morning studies duration (min) Afternoon studies duration (min) Euglycemia Euglycemia a a a a Euglycemia Hypoglycemia a b,c b,c b,c Hypoglycemia Hypoglycemia c c Values are mean SEM. a Afternoon values are significantly increased (P 0.05) as compared with morning euglycemic studies. b Afternoon values are significantly increased (P 0.05) as compared with afternoon hypoglycemia following morning hypoglycemia studies. c Afternoon values are significantly decreased (P 0.05) as compared with morning hypoglycemic studies. decreased by similar levels during morning (62 3to52 2 mm Hg) and afternoon (59 3to52 3 mm Hg) studies. Hyperinsulinemic euglycemic experiments Plasma glucose ( vs mmol/l) and insulin (30 6 vs pmol/l) levels were identical at the start of morning and afternoon euglycemic studies respectively. Steady state plasma glucose values ( vs mmol/l) and insulinemia ( vs pmol/l) were similar during morning and afternoon experiments respectively. Plasma glucagon levels also declined similarly during morning (41 2to33 2 ng/l) and afternoon (37 3to31 2 ng/l). In marked contrast to morning hypoglycemia studies, morning euglycemia improved insulin sensitivity during afternoon studies. Despite equivalent glycemia, glucagon, and insulin levels, glucose infusion rates required to maintain euglycemia were significantly increased in the afternoon ( vs mol/ kg min; P 0.05). Blood glycerol and lactate levels were similar during morning and afternoon euglycemic studies. FFA were significantly (P 0.01) reduced at the start of afternoon ( mol/l) compared with morning studies ( mol/l). Indirect calorimetric values were similar during morning and afternoon experiments as follows: RQ, vs ; glucose disappearance, vs mol/kg min; and lipid disappearance, vs mg/kg min, respectively. Nonoxidative rates of glucose disposal (total glucose infusion rates minus glucose disappearance rates from indirect calorimetry) were significantly increased in the afternoon compared with morning euglycemic studies ( vs mol/ kg min; P 0.01). Discussion This study has determined the effects of moderate antecedent morning hypoglycemia on neuroendocrine and metabolic responses to a second episode (afternoon) of hypoglycemia induced 2 h later. Despite equivalent steady state insulin and glucose levels, epinephrine, norepinephrine, glucagon, cortisol, GH, pancreatic polypeptide, and symptom responses were significantly blunted during the second episode of hypoglycemia. Intermediary metabolism was significantly altered following morning hypoglycemia. Glucose, insulin, FFA, glycerol, -hydroxybutyrate, and lactate levels were significantly increased at the start of afternoon hypoglycemia indicating an insulin-resistant state. Posthypoglycemic insulin resistance was able to offset the blunted neuroendocrine counterregulatory responses by limiting glucose flux during afternoon hypoglycemia. The integrated physiologic effects of a prior episode of hypoglycemia on subsequent same-day hypoglycemia are undetermined. Previous studies have focused on neuroendocrine, cognitive, and symptomatic sequelae of repeated short-term hypoglycemia and provided somewhat conflicting data (1 7). Heller and Cryer (4) determined that an episode of hypoglycemia in normal individuals had minimal effects on counterregulatory responses to hypoglycemia induced 24 h later. Moriarty et al. (26) reported that neuroendocrine responses to moderate hypoglycemia were unaffected by 1 h of immediate prior hypoglycemia. Similarly, Kerr et al. (27) reported that over a 2-h period, two short episodes of intermittent hypoglycemia had no effect on counterregulatory responses. In contrast, Davis et al. (3, 6) have reported that some neuroendocrine responses (glucagon, GH, and cortisol) to a second episode of hypoglycemia were blunted by prior hypoglycemia induced 60 min earlier. Veneman et al. (5), in contrast, demonstrated that one episode of prolonged nocturnal hypoglycemia was sufficient to blunt all major neuroendocrine responses to subsequent morning hypoglycemia. Thus, previous studies have reported a range of differing effects of prior hypoglycemia on subsequent nearterm counterregulatory responses. Our present results demonstrate a substantial, comprehensive blunting of all major counterregulatory responses in the afternoon by morning hypoglycemia. Control experiments determined that morning euglycemia had no effect on modulating neuroendocrine responses during afternoon hypoglycemia. Therefore, we believe that the observed blunting of counterregulatory responses in the afternoon were due to morning hypoglycemia rather than any circadian effects on neuroendocrine release. Thus, our results are conceptually similar to those described by Veneman et al. (5). The percentage blunting of afternoon neuroendocrine responses in this present study ranged from 33 to 55%. Parenthetically, this magnitude of blunting is similar to our previous findings observed after two episodes of antecedent hypoglycemia on next day counterregulatory responses (28). The above previous studies have employed substantially differing experimental designs. Collective analysis of the studies reveals that time seems to play an important role in modulating the effects of antecedent hypoglycemia on subsequent counterregulatory responses. Thus, continuous sequential episodes of antecedent hypoglycemia induced over a period of 2 h are unable to modify counterregulatory re-

7 NEUROENDOCRINE/METABOLIC RESPONSE TO HYPOGLYCEMIA 2049 sponses (26, 27). However, when a period of euglycemia is allowed to supervene, the positive stimulus of hypoglycemia is alleviated and negative signals for down regulation of subsequent neuroendocrine responses are established. The differences in the results of the present study and those of Davis et al. (3, 6) are intriguing. Davis et al. (3, 6) observed no blunting of epinephrine or norepinephrine responses during two episodes of hypoglycemia induced over a 330-min period. The studies of Davis et al. (3, 6) used a 1-h euglycemic period between two episodes of hypoglycemia but maintained continuous hyperinsulinemia throughout the 330-min experiments. Therefore, we would speculate that either 1 h is insufficient for the negative modulation of the sympathetic nervous system by prior hypoglycemia to be manifest and/or continuous hyperinsulinemia may provide a tonic stimulus to the sympathetic nervous system (29) that could offset the negative signal of prior hypoglycemia. Previous studies have documented that hypoglycemia results in a state of insulin resistance (8 10). Multiple mechanisms contribute to this insulin-resistant state with catecholamines, cortisol, and GH implicated in the pathogenesis of this phenomenon (8 10). To date, the acute effects of posthypoglycemic insulin resistance on counterregulatory responses to same-day hypoglycemia have not been determined. At the conclusion of morning hypoglycemia, exogenous insulin was discontinued and the glucose infusion used to maintain the desired hypoglycemic level was increased so that each subject was rapidly restored to euglycemia. However, over a period of min, the glucose infusion was gradually discontinued so that by the start of afternoon hypoglycemia subjects required no exogenous glucose to maintain their plasma glucose. This was in marked contrast to hyperinsulinemic euglycemic control experiments where substantial amounts of glucose ( to mol/kg min) were required at the start of afternoon experiments to maintain plasma glucose at euglycemia. Thus, following morning hypoglycemia, despite the absence of exogenous insulin for 2 h and exogenous glucose for min, plasma glucose and insulin levels were significantly increased relative to the morning indicating an insulinresistant state. Consistent with these findings, plasma FFA, glycerol, and -hydroxybutyrate levels were also increased in the afternoon following morning hypoglycemia indicating increased lipolytic activity (and FFA flux to the liver). Blood lactate levels in the afternoon were also increased following morning hypoglycemia presumably reflecting prolonged catecholamine effects from the morning. Paradoxically, despite blunted neuroendocrine responses, glucose infusion rates and the amount of glucose oxidized during afternoon hypoglycemia were significantly reduced following morning hypoglycemia. Thus, in the short-term, the posthypoglycemic insulin-resistant state was able to compensate via metabolic mechanisms for deficient neuroendocrine responses. This has not been previously reported and may have clinical implications. If similar mechanisms operate in patients with diabetes then the development (or worsening) of insulin resistance after an episode of hypoglycemia may provide a partial defense against subsequent hypoglycemia occurring soon after. The effects of hyperinsulinemia and time on metabolic responses can be determined from the morning hyperinsulinemic euglycemic control studies. In marked contrast to the morning hypoglycemia studies, morning euglycemia substantially increased whole body insulin sensitivity during afternoon studies. Rates of glucose infusion were significantly increased, whereas glycerol and FFA levels were reduced during afternoon hypoglycemia following morning euglycemia (despite preserved neuroendocrine responses) indicating increased insulin sensitivity at the level of skeletal muscle and adipocyte. The contributing mechanisms to this finding seemed to be reduced FFA levels (30) and increased nonoxidative (i.e. glycogen synthesis) glucose metabolism. Hypoglycemic symptoms were significantly reduced in the afternoon following morning hypoglycemia. Although autonomic symptoms were blunted, the majority of the reduced symptom scores were due to diminished neuroglycopenic symptoms. Thus, similar to the neuroendocrine responses, blunted hypoglycemic symptoms can be manifested only 2 h following antecedent hypoglycemia. Recent studies have provided some insight into the mechanisms responsible for diminished neuroendocrine and symptomatic responses following antecedent hypoglycemia. These include antecedent increases in plasma cortisol (31), relatively elevated brain glucose uptake during hypoglycemia (32, 33), and increases in ketone bodies and lactate (34). In this present study, blood -hydroxybutyrate and lactate levels were 2-fold higher at the start of afternoon compared with morning hypoglycemia. These levels are considerably lower than the concentrations of lactate and ketone bodies that have been previously demonstrated to reduce neuroendocrine responses during hypoglycemia (34). Nevertheless, we cannot exclude the possibility that these elevated metabolite levels may have contributed to the blunted neuroendocrine and symptomatic responses occurring during hypoglycemia. In summary, these present results demonstrate that in overnight fasted normal man: 1) a single prolonged episode of moderate hypoglycemia in the morning can significantly blunt neuroendocrine, metabolic and symptom responses to subsequent hypoglycemia induced 2 h later; and 2) prior hypoglycemia induces a state of insulin resistance that in the short-term can partially compensate for diminished neuroendocrine responses by limiting glucose use during subsequent hypoglycemia. Acknowledgments We thank Eric Allen and Pam Venson 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. References 1. Cryer PE Iatrogenic hypoglycemia as a cause of hypoglycemia associated autonomic failure in IDDM. Diabetes. 41: Widom B, Simonson D Intermittent hypoglycemia impairs glucose counterregulation. Diabetes. 41: Davis M, Mellman M, Shamoon H Further defects in counterregulatory responses induced by recurrent hypoglycemia in IDDM. Diabetes. 41: Heller S, Cryer PE Reduced neuroendocrine and symptomatic responses to subsequent hypoglycemia after one episode of hypoglycemia in nondiabetic humans. Diabetes. 40: Veneman T, Mitrakou A, Mokan M, Cryer PE, Gerich J Induction of

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