Differential gender responses to hypoglycemia are due to alterations in CNS drive and not glycemic thresholds

Stephen N. Davis, Chris Shavers, and Fernando Costa

Departments of Medicine, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aims of this study were 1) to determine whether differential glycemic thresholds are the mechanism responsible for the sexual dimorphism present in neuroendocrine responses during hypoglycemia and 2) to define the differences in counterregulatory physiological responses that occur over a range of mild to moderate hypoglycemia in healthy men and women. Fifteen (8 male, 7 female) lean healthy adults underwent four separate randomized 2-h hyperinsulinemic (1.5 mU · kg-1 · min-1) glucose clamp studies at euglycemia (90 mg/dl) or hypoglycemia of 70, 60, or 50 mg/dl. Plasma insulin levels were similar during euglycemic and hypoglycemic studies (91-96 ± 8 µU/ml) in men and women. Hypoglycemia of 70, 60, and 50 mg/dl all resulted in significant increases (P < 0.05, P < 0.01) in epinephrine, glucagon, growth hormone, cortisol, and pancreatic polypeptide levels compared with euglycemic studies in men and women. Plasma norepinephrine levels were increased (P < 0.05) only relative to euglycemic studies at a hypoglycemia of 50 mg/dl. Muscle sympathetic nerve activity (MSNA) increased significantly during hyperinsulinemic-euglycemic control studies. Further elevations of MSNA did not occur until hypoglycemia of 60 mg/dl in both men and women. Plasma epinephrine, glucagon, growth hormone, and pancreatic polypeptide were significantly increased in men compared with women during hypoglycemia of 70, 60, and 50 mg/dl. MSNA, heart rate, and systolic blood pressure responses were also significantly increased in men at hypoglycemia of 60 and 50 mg/dl. In summary, these studies have demonstrated that, in healthy men and women, the glycemic thresholds for activation of epinephrine, glucagon, growth hormone, cortisol, and pancreatic polypeptide occur between 70 and 79 mg/dl. Thresholds for activation of MSNA occur between 60 and 69 mg/dl, whereas norepinephrine is not activated until glycemia is between 50 and 59 mg/dl. We conclude that 1) differential glycemic thresholds are not the cause of the sexual dimorphism present in counterregulatory responses to hypoglycemia; 2) reduced central nervous system efferent input appears to be the mechanism responsible for lowered neuroendocrine responses to hypoglycemia in women; and 3) physiological counterregulatory responses (neuroendocrine, cardiovascular, and autonomic nervous system) are reduced across a broad range of hypoglycemia in healthy women compared with healthy men.

glucose clamp; microneurography; epinephrine; glucagon


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENT STUDIES HAVE IDENTIFIED a significant sexual dimorphism in counterregulatory responses to hypoglycemia (3, 11, 14, 24, 28, 37). Although study designs have differed, the consensus is that a broad spectrum of neuroendocrine responses are reduced in women relative to men (3, 11, 14, 24, 28, 37). The mechanisms responsible for this finding are undetermined. Two previous studies suggested that differential glycemic thresholds for counterregulatory hormone release may explain the reduced neuroendocrine responses in women (14, 37). In other words, plasma glucose values would have to fall to a lower level in women before significant counterregulatory hormone release occurs. This has been challenged by Fanelli et al. (7), who reported similar glycemic thresholds for neuroendocrine responses in healthy men and women using a multi-stepped hypoglycemic experimental design. Thus, at this time, it is controversial whether differences in glycemic thresholds are the mechanism responsible for the sexual dimorphism present in neuroendocrine responses to hypoglycemia.

The experimental methodology used to measure glycemic thresholds has differed in previous studies (4, 7, 27, 29, 32). In general, a protocol involving stepped decrements of glycemia has been compared with a period of euglycemia (either at the start of the experiment or in a separate euglycemic control study) to determine the time when an increase in a particular counterregulatory response occurred.

In the present study, a novel approach has been used to identify glycemic thresholds in a group of healthy men and women. Separate single-level glucose clamp studies of 90, 70, 60, and 50 mg/dl were performed in a randomized fashion. These values represent a euglycemic control and hypoglycemic concentrations previously reported to be glycemic thresholds for release of various neuroendocrine counterregulatory responses. In addition, this experimental design will allow a comprehensive "dose-response" assessment of the differences in counterregulatory physiology present in healthy men and women.

Microneurography was used during the glucose clamp studies so that direct measurement of central sympathetic nervous system (CNS) drive could be obtained. The aims of this study were therefore twofold: 1) to determine whether differential glycemic thresholds are the mechanism responsible for the sexual dimorphism present in counterregulatory responses during hypoglycemia and 2) to determine the integrated physiological responses that occur over a range of mild to moderate hypoglycemia in healthy men and women.


    RESEARCH DESIGN AND METHODS
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

We studied seven healthy females, [age, 27 ± 2 yr; body mass index (BMI), 22.4 ± 0.5 kg/m2; Hb AIC 5.0 ± 0.1% (normal range 4-6.5%)] and eight healthy males (age, 24 ± 2 yr, BMI 22.9 ± 0.6 kg/m2; Hb AIC 4.9 ± 0.1%). None was taking medication or had a family history of diabetes. Each subject had 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 follow their usual weight-maintaining diet for 3 days before each study. Each subject was admitted to the Vanderbilt Clinical Research center at 5:00 PM on the evening before an experiment. All subjects were studied after an overnight 10-h fast.

Experimental Design

Glucose clamp studies. Subjects attended four separate experiments separated by >= 2 mo. The order of the four experiments was randomized and was performed in a single-blind fashion. Studies in women were performed during the follicular phase of a menstrual cycle. On the morning of each study, after an overnight fast, two intravenous 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 (1). 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).

After insertion of the venous cannulae, a period of 90 min was allowed to elapse, followed by a 30-min basal control period and a 120-min hyperinsulinemic experimental period of either 90, 70, 60, or 50 mg/dl. At time 0, a primed continuous infusion of insulin (33) was administered at a rate of 1.5 mU · kg-1 · min-1 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 (5, 13). Potassium chloride (20 mmol/l) was added to the glucose infusate in each study. A separate group of six healthy males (age, 26 ± 1 yr; BMI, 22 ± 0.4; Hb AIC 4.6 ± 0.2%) underwent a single hyperinsulinemic study at 80 mg/dl plasma glucose as described above.

Direct measurement of muscle sympathetic nerve activity. Microneurographic activity was recorded from the peroneal nerve at the level of the fibular head (35). The approximate location of this nerve was determined by transdermal electrical stimulation (10-60 V, 0.01 ms duration), which produces painless muscle contraction of the foot. After this, a reference tungsten electrode with a shaft diameter of 200 µm was placed subcutaneously. 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 proprioceptive afferent signals, whereas cutaneous stimulation by slight 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 studies, because this has been demonstrated to reflect increased sympathetic activity during insulin-induced hypoglycemia (17), 2-deoxyglucose-induced neuroglycopenia (16), and hyperinsulinemic euglycemia in normal humans (6).

Sympathetic nerve activity is expressed as bursts per minute. Measurements of MSNA were made from the original tracings using a digitizer tablet (HIPAD, Houston Instruments, Austin, TX) coupled to Sigma Scan Software (Jandel Scientific, Coite Modena, CA) in a microcomputer.

Analytical methods. The collection and processing of blood samples have been described elsewhere (10). Plasma glucose concentrations were measured in triplicate using the glucose oxidase method with a glucose analyzer (Beckman, Fullerton, CA). Glucagon was measured according to the method of Aguilar-Parada et al. (2) with an interassay coefficient of variation (CV) of 15%. Insulin was measured as described previously (36) with an interassay CV of 11%. Catecholamines were determined by HPLC (9) with an interassay CV of 12% for epinephrine and 12% 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 by use of the Clinical Assays Gamma Coat RIA kit with an interassay CV of 6%. Growth hormone was determined by RIA (23) with a CV of 8%. Pancreatic polypeptide was measured by RIA according to the method of Hagopian et al. (20) with an interassay CV of 8%. Lactate, glycerol, alanine, and 3-hydroxybutyrate were measured on deproteinized whole blood according to the method of Lloyd et al. (26). Nonesterified fatty acids (NEFA) were measured with the use of the WAKO kit, adapted for use on a centrifugal analyzer (22).

Blood for hormones and intermediary metabolites were drawn twice during the control period and every 15 min during the experimental period. Cardiovascular parameters (pulse, systolic, diastolic, and mean arterial pressures) were measured noninvasively by a Dinamap (Critikon, Tampa, FL) every 10 min throughout each 300-min study. MSNA was measured continuously throughout each 300-min study.

Materials. 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. Between-group analysis of men and women was analyzed by standard parametric two-way ANOVA with a repeated measures design. This was coupled with Duncan's post hoc test to delineate the time when statistical significance was reached. A P value of < 0.05 indicated significant difference. Glycemic thresholds for various parameters were determined by two methods. 1) As previously described (32), the glycemic threshold for a given parameter was the glucose level when the value of a parameter exceeded the 95% confidence limit observed for that parameter at the corresponding time point in euglycemic control experiments. 2) One-way ANOVA with a repeated-measures design was applied to each glycemic level to determine whether a significant increase in a given parameter occurred relative to baseline. Duncan's post hoc test was then applied to determine the time point when statistical significance was reached.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucose and Insulin Levels

Steady-state plasma glucose levels were equivalent in men (93 ± 2, 70 ± 2, 60 ± 2, and 53 ± 2 mg/dl) and women (92 ± 2, 70 ± 2, 61 ± 2, and 53 ± 2 mg/dl) during both series of glucose clamps (Fig. 1). Insulin levels were also similar in men (96 ± 8 µU/ml) and women (91 ± 9 µU/ml) during all experiments (Fig. 1).


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Fig. 1.   Arterialized plasma insulin and glucose levels during euglycemic and hypoglycemic clamps in overnight-fasted healthy men and women.

Neuroendocrine Levels

Epinephrine. Epinephrine levels remained similar to baseline during euglycemic control studies in men and women (Fig. 2 and Table 1). By the final 30 min, hypoglycemia of 70 mg/dl had induced significant increases in epinephrine levels in men and women. The increases in men (44 ± 9 to 217 ± 61 pg/ml) were much greater (P < 0.01) compared with women (38 ± 8 to 75 ± 21 pg/ml). Epinephrine levels increased in a dose-dependent manner during hypoglycemia of 60 and 50 mg/dl in men (611 ± 71 and 934 ± 110 pg/ml, respectively) and women (216 ± 65 and 628 ± 148 pg/ml, respectively). Epinephrine values were significantly greater (P < 0.01) in men compared with women at all hypoglycemic levels.


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Fig. 2.   Effects of peripherally infused insulin (1.5 mU · kg-1 · min-1), in the presence of euglycemia (90 mg/dl) and hypoglycemia (70, 60, 50 mg/dl) on incremental from baseline responses of epinephrine, norepinephrine, and muscle sympathetic nerve activity (MSNA) in overnight-fasted healthy men and women. ** Plasma epinephrine responses significantly increased (P < 0.01) in men vs. women at hypoglycemia of 70, 60, and 50 mg/dl. Epinephrine responses in men and women significantly increased (P < 0.05) during all hypoglycemic levels vs. euglycemia. Plasma norepinephrine responses significantly increased (P < 0.05) in men and women during hypoglycemia of 50 mg/dl vs. euglycemia. ** MSNA responses significantly increased (P < 0.01) in men vs. women during hypoglycemia of 60 and 50 mg/dl. MSNA responses in men and women significantly increased (P < 0.05) during hypoglycemia of 60 and 50 mg/dl vs. euglycemia.


                              
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Table 1.   Effects of hyperinsulinemic (1.5 mU · kg-1 · min-1) euglycemia or hypoglycemia of 70, 60, 50 mg/dl on neuroendocrine and MSNA responses in overnight-fasted healthy men and women

Norepinephrine. Plasma norepinephrine levels increased significantly (P < 0.05) during the hyperinsulinemic euglycemic control studies in men (163 ± 15 to 225 ± 20 pg/ml) and women (178 ± 20 to 221 ± 21 pg/ml). Hypoglycemia of 70 and 60 mg/dl produced similar norepinephrine levels in men and women that were not different from euglycemic controls. Only hypoglycemia of 50 mg/dl produced significantly increased responses compared with responses in control studies. Norepinephrine values in men (165 ± 10 to 328 ± 20 pg/ml) and women (153 ± 14 to 307 ± 38 pg/ml) were increased similarly (Fig. 2, Table 1).

Glucagon. Glucagon levels (Fig. 3 and Table 1) were suppressed (P < 0.05) during euglycemic control studies in men (78 ± 10 to 59 ± 3 ng/l) and women (64 ± 10 to 47 ± 7 ng/l). During hypoglycemia of 70 mg/dl, glucagon levels increased significantly in men (80 ± 9 to 132 ± 28 ng/l, P < 0.01) but remained similar to baseline in women (63 ± 7 to 69 ± 12 ng/l). However, glucagon levels in women during the 70-mg/dl studies were significantly increased compared with control experiments (69 ± 12 vs. 47 ± 8 ng/l, P < 0.05). Hypoglycemia of 60 and 50 mg/dl in men resulted in further, similarly increased levels of glucagon (276 ± 44 and 253 ± 35 pg/ml, respectively). In women, hypoglycemia of 60 and 50 mg/dl produced modest stepwise increases in glucagon (88 ± 10 and 109 ± 23 pg/ml, respectively). At all hypoglycemic levels, glucagon values were at least twofold greater (P < 0.01) in men relative to women.


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Fig. 3.   Effects of peripherally infused insulin (1.5 mU · kg-1 · min-1) in the presence of euglycemia (90 mg/dl) and hypoglycemia (70, 60, 50 mg/dl) on incremental from baseline responses of plasma cortisol and glucagon. ** Cortisol responses significantly increased (P < 0.01) in men during hypoglycemia of 60 mg/dl vs. women. Plasma cortisol responses in men and women significantly increased (P < 0.01) at all hypoglycemic levels vs. euglycemia. ** Plasma glucagon responses significantly increased (P < 0.01) in men vs. women at hypoglycemia of 70, 60, and 50 mg/dl. Glucagon responses in men and women significantly increased (P < 0.01) at all hypoglycemic levels vs. euglycemia.

Cortisol. Plasma cortisol levels (Fig. 3 and Table 1) remained similar to baseline during euglycemic controls in men and women. Compared with euglycemic control studies, hypoglycemia of 70 mg/dl produced similar significant increases of cortisol in men (8 ± 1 to 13 ± 1 µg/dl) and women (9 ± 1 to 13 ± 1 µg/dl). Cortisol levels increased further during hypoglycemia of 60 mg/dl in men but not in women. Hypoglycemia of 50 mg/dl produced similar elevations of cortisol in men and in women.

Growth hormone. Growth hormone levels remained similar to baseline during euglycemic control studies in men and women (Fig. 4 and Table 1). Compared with control studies, hypoglycemia of 70 mg/dl produced significant (P < 0.05) increases in growth hormone in men (2 ± 1 to 22 ± 5 ng/ml) and women (4 ± 1 to 15 ± 5 ng/ml). Hypoglycemia of 60 and 50 mg/dl produced further increases in growth hormone in men and women. Growth hormone levels were significantly greater (P < 0.01) in men at 60 and 50 mg/dl compared with women.


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Fig. 4.   Effects of peripherally infused insulin (1.5 mU · kg-1 · min-1) in the presence of euglycemia (90 mg/dl) and hypoglycemia (70, 60, 50 mg/dl) on incremental from baseline responses of plasma growth hormone and pancreatic polypeptide. Growth hormone responses significantly increased (* P < 0.05 ** P < 0.01) in men during hypoglycemia of 70, 60, and 50 mg/dl vs. women. Plasma growth hormone responses significantly increased (P < 0.01) in men and women at all hypoglycemic levels vs. euglycemia. Plasma pancreatic polypeptide responses significantly increased (* P < 0.05 ** P < 0.01) in men during hypoglycemia of 70, 60, and 50 mg/dl vs. women. Plasma pancreatic polypeptide responses significantly increased (P < 0.05-P < 0.01) in men and women at all hypoglycemic levels vs. euglycemia.

Pancreatic polypeptide. Pancreatic polypeptide levels (Fig. 4 and Table 1) were similarly suppressed during euglycemic controls in men and women. Compared with control studies, hypoglycemia of 70 mg/dl produced significant increases (P < 0.05) in pancreatic polypeptide levels in men and women. The elevations in men (170 ± 30 to 337 ± 89 pg/ml) were significantly greater (P < 0.01) compared with women (131 ± 40 to 181 ± 61 pg/ml). Hypoglycemia of 60 and 50 mg/dl produced stepwise greater responses in pancreatic polypeptide in men and women. However, pancreatic polypeptide levels were significantly higher in men compared with women (P < 0.05) at all hypoglycemic levels.

MSNA

MSNA increased (Table 1 and Fig. 2) by a small but significant amount (P < 0.05) during euglycemic controls in men (+4 ± 1 bursts/min) and in women (+3 ± 1 bursts/min). Hypoglycemia of 70 mg/dl produced similar increases in men and women and was not different from control studies. Hypoglycemia of 60 mg/dl produced significantly increased MSNA responses compared with euglycemic controls in men (+14 ± 3 vs. +4 ± 1 bursts/min) and women (+7 ± 2 vs. +3 ± 1 bursts/min). MSNA responses were significantly increased (P < 0.01) during hypoglycemia of 60 and 50 mg/dl in men relative to women.

Metabolic Responses

Glucose infusion rates used to maintain euglycemia during control studies were equivalent in men and women. Increasing levels of hypoglycemia resulted in significant reductions in glucose infusion rates in men and women (Table 2). However, at each hypoglycemic level commensurate with the increased neuroendocrine response, the glucose infusion rates were significantly (P < 0.01) reduced in men compared with women.

                              
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Table 2.   Glucose infusion rates required to maintain euglycemia or hypoglycemia of 70, 60, and 50 mg/dl in the presence of peripheral insulin infusion in overnight-fasted healthy men and women

Blood glycerol and plasma NEFA levels were significantly suppressed during euglycemic control studies in men and women (Table 3). Hypoglycemia of 70 mg/dl in women resulted in similar glycerol and NEFA levels compared with euglycemic controls. Hypoglycemia of 60 and 50 mg/dl produced stepwise increases of glycerol and NEFA in men and women. Glycerol levels at baseline and at the end of the euglycemic, 60-, and 50-mg/dl studies were significantly higher (P < 0.05) in women compared with men.

                              
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Table 3.   Effects of hyperinsulinemic euglycemia or hypoglycemia of 70, 60, 50 mg/dl on intermediary metabolites in overnight-fasted healthy men and women

Blood lactate levels in men increased progressively with each deepening hypoglycemic level (Table 3). Lactate levels in women increased only from euglycemia to the hypoglycemia of 60 mg/dl. Blood lactate levels during hypoglycemia of 50 mg/dl were significantly increased in men relative to women. beta -Hydroxybutyrate levels were significantly and similarly suppressed during euglycemic and hypoglycemic clamp studies in men and women.

Cardiovascular Responses

Heart rate remained unchanged during euglycemic control studies in men and women (Table 4). During hypoglycemia of 70 mg/dl, heart rate increased significantly (P < 0.05) in men and women, although there was a greater response in men (+9 ± 2 vs. +5 ± 1 beats/min, P < 0.05). Hypoglycemia of 60 and 50 mg/dl in men produced further stepwise increases in heart rate. In women, deeper hypoglycemia of 60 and 50 mg/dl did not increase heart rate relative to milder hypoglycemia of 70 mg/dl.

                              
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Table 4.   Effects of hyperinsulinemic euglycemia or hypoglycemia of 70, 60, 50 mg/dl on cardiovascular responses in overnight-fasted healthy men and women

Systolic blood pressure did not change from baseline during control studies in men and women. Increasing depth of hypoglycemia in men produced progressively greater systolic blood pressure values. In women, hypoglycemia of 70 and 60 mg/dl did not increase blood pressure relative to control studies. Hypoglycemia of 50 mg/dl produced a small increase in systolic blood pressure that was significantly reduced compared with men (+4 ± 1 vs. +12 ± 2 mmHg, P < 0.05).

Neuroendocrine and MSNA Responses

Neuroendocrine responses at plasma glucose of 80 mg/dl. Six additional male subjects were studied during hyperinsulinemic-clamped glycemia of 80 mg/dl. Epinephrine (41 ± 6 to 50 ± 10 pg/ml), cortisol (10 ± 1 to 10 ± 2 µg/dl), and growth hormone (1.5 ± 0.3 to 1.8 ± 0.7 ng/ml) remained unchanged relative to baseline. Glucagon (65 ± 6 to 58 ± 6 ng/l) and pancreatic polypeptide (148 ± 35 to 93 ± 34 pg/ml) levels tended to fall by the final 30 min of the 2-h studies. Plasma norepinephrine levels increased significantly (204 ± 29 to 275 ± 40 pg/ml, P < 0.05) compared with baseline. The neuroendocrine responses obtained from the 80-mg/dl experiments were not significantly different from those obtained during the euglycemic control studies.

Quantitative estimations of glycemic thresholds for neuroendocrine and MSNA responses. Values for each parameter from the final 30 min of the glucose clamps (90, 80, 70, 60, and 50 mg/dl) were plotted in a linear or sigmoid dose-response fashion. The quantitative values are presented in Table 5.

                              
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Table 5.   Effects of hyperinsulinemic hypoglycemia on plasma glycemic thresholds (mg/dl) for activation of neuroendocrine and muscle sympathetic nerve activity counterregulatory responses

Glycemic thresholds were similar in men and women. Glycemic thresholds for neuroendocrine responses (exception norepinephrine) were lower when plotted in a sigmoid dose-response manner and ranged from 71 ± 2 to 78 ± 2 mg/dl, depending on individual counterregulatory hormones. Depending on the method of quantification, glycemic thresholds for MSNA ranged from 61 ± 2 to 67 ± 2 mg/dl and from 51 ± 2 to 56 ± 1 mg/dl for norepinephrine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goals of this study were 1) to obtain a comprehensive assessment of gender-related differences in counterregulatory physiology occurring during hypoglycemia and 2) to determine whether differential glycemic thresholds are the mechanism responsible for the sexual dimorphism present in counterregulatory responses to hypoglycemia in healthy men and women. These present results have demonstrated that the majority of neuroendocrine responses to hypoglycemia are activated at or above a glucose level of 70 mg/dl in healthy men and women. Diminished epinephrine, pancreatic polypeptide, growth hormone, and MSNA responses in women during hypoglycemia of 70, 60, and 50 mg/dl indicate reduced CNS efferent input as a significant mechanism responsible for the gender-related differences in counterregulatory physiology present in healthy men and women.

In this present study, neuroendocrine, metabolic, and cardiovascular responses have been determined in healthy men and women during four separate 2-h hyperinsulinemic studies at euglycemia and at hypoglycemic levels of 70, 60, and 50 mg/dl. The euglycemic studies provide controls for the independent effects of insulin and time. Hyperinsulinemic euglycemia had no effect on epinephrine, growth hormone, or cortisol levels. Glucagon and pancreatic polypeptide levels were significantly suppressed during euglycemic controls. Norepinephrine values, on the other hand, increased significantly in men and women during euglycemic controls. Hypoglycemia of 70 mg/dl produced significant increases of epinephrine, growth hormone, and cortisol in men and women. Increases of epinephrine and growth hormone were greater in men compared with women. Glucagon and pancreatic polypeptide responses increased significantly in men during hypoglycemia of 70 mg/dl but remained similar to baseline in women. However, the lack of a fall in glucagon and pancreatic polypeptide levels represented a significant increase compared with the suppression of these parameters that occurred during control studies.

Glycemic thresholds for norepinephrine and MSNA are more difficult to interpret. Insulin produces dose-dependent increases of norepinephrine and MSNA, even under euglycemic conditions (6). Thus, unlike the other counterregulatory parameters examined in this study, the glycemic threshold of norepinephrine and MSNA involved an additional secondary hypoglycemic signal to overcome the stimulus of insulin per se. At face value, our results indicate that the threshold for MSNA (61-67 mg/dl) is intermediate between those of epinephrine (72-78 mg/dl) and norepinephrine (51-56 mg/dl). However, we believe that the threshold values for MSNA and norepinephrine may have been strongly influenced (i.e., underestimated) by the prevailing insulinemia. Thus, if a very low dose of insulin has been used to produce hypoglycemia that had minimal effects on stimulating the sympathetic nervous system, then we speculate that the thresholds for both MSNA and norepinephrine could be similar to those of the other counterregulatory hormones. However, we would like to stress that glycemic thresholds for norepinephrine and MSNA occurred at similar glucose levels in men and women.

In an attempt to determine whether neuroendocrine activation can occur at glycemia >70 mg/dl, six additional male subjects were studied during 80-mg/dl glucose clamps. Men were studied because their counterregulatory responses at 70 mg/dl were greater than the more modest values occurring in women. We therefore reasoned that, if 80 mg/dl were to have any effects on neuroendocrine responses, it would be observed only in the context of a larger experimental signal in men. However, the results of these experiments demonstrate that hyperinsulinemic studies at 80 mg/dl produced no significant activation of neuroendocrine responses.

We would, therefore, interpret our present results to indicate that, with the exception of norepinephrine, the glycemic thresholds for neuroendocrine release in men and women lie between glucose levels of 71 and 78 mg/dl. Our present results are consistent with most (8, 12, 18, 27, 29, 32), but not all (4, 31), previous studies that concluded that glycemic thresholds for counterregulatory hormone release occurs somewhere between 63 and 80 mg/dl. The finding that the glycemic threshold for cortisol release is close to 70 mg/dl is supported by some (8, 12), but not all, previous studies (4, 29, 31, 32).

We believe that the controversy in the literature arises for two reasons. First, similar to norepinephrine, insulin can stimulate cortisol release, even under euglycemic conditions (7). Therefore, the determination of a glycemic threshold for cortisol under hyperinsulinemic conditions is complex, because an additional secondary signal of deeper hypoglycemia is required to overcome the stimulus of insulin per se. Second, plasma cortisol levels require >60 min to reach steady state during a single-level hypoglycemic study. Thus any study that uses a stepped decrement in glucose design with only 30- to 45-min glycemic plateaus may underestimate the threshold for cortisol, because the hormone would not have had sufficient time to reach maximal steady-state level. Therefore, this present study (incorporating a single hypoglycemic level of 2 h), in reporting that the glycemic threshold for cortisol is similar to other counterregulatory hormones (8, 12), is consistent with previous studies that have maintained decrements in plasma glucose for >1 h.

Epinephrine, glucagon, growth hormone, and pancreatic polypeptide responses were greater at each hypoglycemic level in men compared with women. MSNA responses were greater in men during hypoglycemia of 60 and 50 mg/dl relative to women. In fact, increases in MSNA activity during hypoglycemia in women is markedly attenuated and sime 50% of this increment is due to insulin rather than hypoglycemia per se. This demonstrates that the sexual dimorphism in counterregulatory responses extends through the range of mild-to-moderate hypoglycemia.

The results from this present study are conceptually similar to those of Fanelli et al. (18) who employed a stepped hypoglycemia design. Fanelli et al. reported that, with the exception of norepinephrine, glycemic thresholds for counterregulatory hormones ranged from 65 to 69 ± 1 mg/dl in men and women. Our present results also demonstrate that, with the exception of norepinephrine, glycemic thresholds for neuroendocrine activation lie between 71 and 78 mg/dl. The one notable difference between our study and that of Fanelli et al. is in the glucose infusion rates required to maintain hypoglycemia. Despite reduced counterregulatory responses in women, Fanelli et al. reported that the glucose infusion rates required to maintain hypoglycemia in women were significantly reduced compared with men, thus concluding that, at least under hypoglycemic conditions, women were less insulin sensitive than men. In the present study, we found the glucose infusion rates used during the euglycemic hyperinsulinemic controls to be equivalent in men and women. Additionally, commensurate with the reduced neuroendocrine responses, glucose infusion rates were significantly increased at each hypoglycemic level in women compared with men. Thus, in the present study, we could not determine any difference in insulin sensitivity between men and women. Again, we believe that the difference between the two studies is due to differing experimental designs. In the present study, a single prolonged level will negate the possibility of "non-steady-state effects" that could be present in shorter multiple glycemic plateaus.

From the above, it is clear that glycemic thresholds for all major neuroendocrine counterregulatory responses and MSNA are similar in men and women. Therefore, differential glycemic thresholds cannot explain the reduced counterregulatory responses to hypoglycemia present in women. Previous studies have demonstrated that nonglucose metabolites (i.e., lactate and ketone bodies) can be used acutely by the brain as fuel during hypoglycemia and downregulate neuroendocrine responses (19, 34). In the present study, however, there was either no difference in the levels of these metabolites in men and women or, if anything, they (lactate) were higher in males. The reduced epinephrine, growth hormone, pancreatic polypeptide, and MSNA responses all indicate a reduced central (i.e., brain) efferent response during hypoglycemia in women. Glucagon release was also substantially lower in women. Control of glucagon secretion during insulin-induced hypoglycemia is complex. Stimulatory signals include direct alpha -cell sensing and autonomic nervous system input (21, 30), whereas insulin can negatively inhibit secretion (25). Therefore, it is difficult to determine whether it is reduced stimulatory or increased inhibitory mechanisms that are responsible for decreased glucagon secretion in women. However, it is interesting to note that, during the hyperinsulinemic euglycemic control studies, glucagon was suppressed similarly in men and women. Thus we do not believe that the alpha -cells in women are more susceptible to the suppressive effects of insulin compared with men. Interestingly, cortisol levels in women were similar to those in men in two of the three hypoglycemic levels (70 and 50 mg/dl). ACTH levels were measured during the 50 mg/dl studies and revealed no difference in men (70 ± 20 pg/ml) and women (73 ± 21 pg/ml). We would, therefore, speculate that central regulation of cortisol in adults (a hormone with additional nonglucoregulatory functions) is different from the more strictly defined "metabolic" hormones epinephrine and growth hormone. The cause of the reduced hypoglycemic drive needs further study. Possibilities include 1) signals from peripheral sites such as adipose tissue and/or liver (15) or 2) direct CNS effects of circulating hormones or neurotransmitters.

In summary, these present studies have demonstrated that the glycemic thresholds for activation of neuroendocrine and sympathetic nervous system responses during hypoglycemia do not differ in healthy men and women. During hyperinsulinemic conditions in the mid-physiological range, thresholds for activation of epinephrine, glucagon, growth hormone, cortisol, and pancreatic polypeptide occur between 71 and 78 mg/dl. Thresholds for activation of MSNA occur between 61 and 67 mg/dl, whereas norepinephrine is activated by a hypoglycemia of between 51 and 56 mg/dl. Despite similar glycemic thresholds, counterregulatory responses are reduced across the range of mild to moderate hypoglycemia. We conclude that 1) differential glycemic thresholds are not the cause of the sexual dimorphism present in counterregulatory responses to hypoglycemia; 2) reduced CNS efferent input appears to be the mechanism responsible for lowered neuroendocrine responses to hypoglycemia in women; and 3) physiological responses (neuroendocrine, cardiovascular, and autonomic nervous system) are reduced across a broad range of hypoglycemia in healthy women compared with healthy men.


    ACKNOWLEDGEMENTS

We thank Eric Allen, Pam Venson, and Wanda Snead for expert technical assistance. We are also grateful for the superb care and help provided by the nursing staff of the Vanderbilt General Clinical Research Center.


    FOOTNOTES

This work was supported by a research grant from the Juvenile Diabetes Foundation International, National Institutes of Health Grant RO1-DK-45369, Diabetes Research and Training Center Grant 5P60-AM-20593, and Clinical Research Center Grant MO1-RR-00095.

Address for reprint requests and other correspondence: S. N. Davis, 712 MRBII, Division of Diabetes and Endocrinology, Vanderbilt Medical School, Nashville, TN 37232 (E-mail: steve.davis{at}mcmail.vanderbilt.edu).

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.

Received 4 October 1999; accepted in final form 12 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
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