HYPOGLYCEMIA is a major adverse effect of intensive

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1 X/99/$03.00/0 Vol. 84, No. 5 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society Protective Effect of Insulin against Hypoglycemia- Associated Counterregulatory Failure BERND FRUEHWALD-SCHULTES, WERNER KERN, EVA DEININGER, PETER WELLHOENER, WOLFGANG KERNER, JAN BORN, HORST L. FEHM, AND ACHIM PETERS Departments of Internal Medicine I (B.F.-S., W.K., W.B., P.W., H.L.F., A.P.) and Clinical Neuroendocrinology (J.B.), University of Luebeck, D Luebeck, and the Department of Diabetes and Metabolism, Klinikum Karlsburg (W.K.), D Karlsburg, Germany ABSTRACT Antecedent hypoglycemic episodes reduce the counterregulatory neuroendocrine response to hypoglycemia. The role of insulin in the mechanism responsible for the antecedent hypoglycemia causing subsequent counterregulatory failure has not been elucidated. We performed antecedent hypoglycemic clamps (56 mg/dl) lasting 2 h with differing degrees of hyperinsulinemia, which were followed by 6-h stepwise hypoglycemic clamps ( mg/dl) on the next day. Experiments were carried out in 30 young, healthy men. Fifteen of these subjects were tested on 2 occasions. On 1 occasion the antecedent hypoglycemia was induced by insulin infusion at a rate of 1.5 mu/min kg (low insulin-ante-hypo); on the other occasion the insulin infusion rate was 15.0 mu/min kg (high insulin-ante-hypo). Both sessions were separated by at least 4 weeks, and their order was balanced HYPOGLYCEMIA is a major adverse effect of intensive insulin therapy (1). Many patients with well controlled type 1 diabetes have a diminished counterregulatory hormone response to hypoglycemia (2, 3). Antecedent hypoglycemia was shown to reduce the counterregulatory hormone response to subsequent hypoglycemia (4 10). The mechanisms mediating this effect remained obscure. One proposed mechanism is an adaptation of the brain to hypoglycemia based on an increased proportional extraction of glucose during hypoglycemia (11). Such an improved glucose uptake into the brain has been demonstrated after prolonged hypoglycemia in rats (12) and humans (13) and could be based on an increased expression of glucose transporter (GLUT1) molecules (14). Another mechanism responsible for the antecedent hypoglycemia causing subsequent counterregulatory failure is suggested by findings of Davis et al. (15). They demonstrated that physiological increases in cortisol levels after infusion of hydrocortisone can mimic the effects of antecedent hypoglycemia on subsequent counterregulation. Davis et al. concluded that increased plasma cortisol is the factor causing autonomic counterregulatory failure after antecedent hypoglycemia (15). However, the role of insulin in the pathogenesis of deficient counterregulation due to antecedent hypoglycemia has Received September 16, Revision received December 4, Accepted February 2, Address all correspondence and requests for reprints to: Bernd Fruehwald-Schultes, M.D., Medical University Luebeck, Department of Internal Medicine I, Ratzeburger Allee 160, D Luebeck, Germany. fruehwal@kfg.mu-luebeck.de. across subjects. The remaining 15 subjects (control group) received the same stepwise hypoglycemic clamp as the other subjects, but without antecedent hypoglycemia. During the stepwise hypoglycemic clamp, the counterregulatory increases in ACTH, cortisol, and norepinephrine were significantly blunted after the low insulin-antehypo (P 0.01, P 0.05, and P 0.05, respectively) but not after the high insulin-ante-hypo (P 0.12, P 0.92, and P 0.19, respectively) compared to that in the control group. The cortisol, norepinephrine, and glucagon responses were greater after the high than after the low insulin-ante-hypo (all P 0.05). In conclusion, the present study clearly demonstrates that even a single episode of mild hypoglycemia reduces neuroendocrine counterregulation h later. Insulin has a moderate protective effect on subsequent counterregulation. (J Clin Endocrinol Metab 84: , 1999) not been studied. The aim of this study, therefore, was to determine whether and to what extent the level of insulin during antecedent hypoglycemia influences counterregulation during subsequent hypoglycemia. Subjects Subjects and Methods Thirty young, healthy men participated in the experiments (age, 26 1 yr; body mass index, kg/m 2 ). Exclusion criteria were chronic or acute illness, current medication of any kind, smoking, alcohol or drug abuse, obesity, and diabetes or hypertension in first degree relatives. Each volunteer gave written informed consent, and the study was approved by the local ethics committee. Study design Stepwise hypoglycemic clamp experiments (stepwise hypoglycemia) lasting 6 h were performed in all 30 subjects. Fifteen of the subjects, randomly selected, additionally underwent an antecedent hypoglycemic clamp lasting 2.5 h on the day preceding the stepwise hypoglycemia (ante-hypo), whereas the other 15 subjects did not (control group). The 15 subjects receiving the ante-hypo were studied twice, so that they had a total of 2 antecedent hypoglycemic and 2 stepwise hypoglycemic clamps. On 1 of these occasions the antecedent hypoglycemia was induced by an insulin infusion at a rate of 1.5 mu/min kg (low insulinante-hypo); on the other occasion the infusion rate was 15.0 mu/min kg (high insulin-ante-hypo). The sequence of the 2 antecedent hypoglycemic clamps was random, and a 4-week recovery period lay between the 2 testing occasions for a subject. Antecedent hypoglycemic clamp On the day of the antecedent hypoglycemic clamp, the subjects reported to the medical research unit at 1330 h. The subjects were informed 1551

2 1552 FRUEHWALD-SCHULTES ET AL. JCE&M 1999 Vol 84 No 5 FIG. 1. Mean (SEM) plasma glucose concentrations (top) and serum insulin concentrations (bottom) during the antecedent (left) and subsequent stepwise (right) hypoglycemic clamp. The SEMs of serum insulin concentrations were smaller than the sizes of the symbols. The control group (open triangle, dotted lines) did not include antecedent hypoglycemia. Black triangle (thin lines), Low insulin-ante-hypo protocol; black square (thick lines), high insulin-ante-hypo protocol. not to have breakfast on this day and to abstain from eating until the end of the clamp. The experiments took place in a sound-attenuated room with the subjects sitting with their trunk in an almost upright position (about 60 ) and their legs in a horizontal position on the bed. A cannula was inserted into a vein on the back of the hand, which was placed in a heated box (50 55 C) to obtain arterialized venous blood. A second cannula was inserted into an antecubital vein of the contralateral arm. Both cannulas were connected to long thin tubes, which enabled blood sampling and adjustment of the rate of dextrose infusion from an adjacent room without being noticed by the subject. At 1400 h, infusion of insulin (H-insulin, Hoechst, Frankfurt, Germany) began at a continuous rate of either 1.5 or 15.0 mu/min kg, respectively, depending upon the protocol. Plasma glucose levels were measured (glucose analyzer, Beckman Coulter, Inc. Munich, Germany) every 5 min, and a variable infusion of 20% dextrose solution was adjusted so that plasma glucose levels were held constant at approximately 56 mg/dl. Blood samples for determination of serum levels of insulin and cortisol were collected every 30 min during the 2.5 h of the hypoglycemic clamp. Stepwise hypoglycemic clamp On the day of the stepwise hypoglycemic clamp, all subjects reported to the medical research unit at 0800 h after an overnight fast of at least 10 h. The setting of the stepwise hypoglycemic clamps was the same as the setting of the antecedent hypoglycemic clamps. After a 1-h baseline period, insulin was infused at a continuous rate of 1.5 mu/min kg for the next 6 h. Arterialized blood was drawn at 5-min intervals to measure the plasma glucose concentration. A 20% dextrose solution was simultaneously infused at a variable rate to control plasma glucose levels. Plasma glucose levels were reduced in a stepwise manner to achieve four respective plateaus of approximately 76, 66, 56, and 46 mg/dl. Each plateau was maintained for a 45-min period, and the next lower plateau was induced gradually within the next 45 min. Measurements Blood samples were collected every 30 min and immediately centrifuged, and the supernatants were stored at 24 C until assay. Serum insulin, cortisol, GH, and glucagon and plasma ACTH, epinephrine, and norepinephrine were measured as previously described (16). A semiquantitative symptom questionnaire was administered every 15 min. Subjects scored from 0 (none) to 4 (severe) on each of the following symptoms: tremor, inner restlessness, hunger, palpitations, sweating, tingling around lips, nervousness, weakness, passivity, warmth, blurred vision, dizziness, headache, and drowsiness. Consistent with the categorization used by previous investigators (17), the first seven symptoms where were considered autonomic, and the other seven were considered neuroglycopenic. The sum of each of these constitutes the symptom score. Statistical methods All values are presented as the mean sem. Statistical analysis included paired and unpaired Student s t test, Fisher s exact test, and McNemar s test for nonparametric comparisons and determination of Pearson s correlation coefficients. P 0.05 was considered significant.

3 INSULIN AND HYPOGLYCEMIC COUNTERREGULATION 1553 Results Antecedent hypoglycemic clamp During both the high and low rates of insulin infusion, plasma glucose concentrations decreased from about 68 to 56 mg/dl within the first 30 min and were maintained at this level until the end of the clamp (Fig. 1). Mean serum insulin concentrations were approximately 40-fold higher ( vs mu/ml; P ; Fig. 1), whereas the average dextrose infusion rate was only 1.8-fold higher [ vs mg/kg min (dextrose/body weight time); P ] during the high than during the low insulin antecedent hypoglycemia. Both the low and high insulin antecedent hypoglycemic clamps induced a distinct rise in cortisol (both P ; Fig. 2). However, the rise in cortisol developed earlier and was more pronounced during the high than during the low rate of insulin infusion. Accordingly, the mean serum cortisol concentration during the last 2 h of the clamp was greater in the high than in the low insulin-ante-hypo ( vs g/dl; P 0.05). Stepwise hypoglycemic clamp After starting the insulin infusion, plasma glucose levels decreased, and serum insulin levels increased in the same manner in all protocols (Fig. 1). Baseline levels and changes in all measurements during the stepwise hypoglycemia are listed in Table 1. At baseline, plasma ACTH levels were lower after both the low and the high insulin-ante-hypo than in the control group (both P 0.05). During the stepwise hypoglycemic clamp, plasma ACTH increased in all protocols (all P ; Fig. 2). The ACTH increase was significantly lower after the low insulin-ante-hypo than in the control group (P 0.01), whereas the difference between values in the high insulinante-hypo protocol and the control group failed to reach significance (P 0.12). During the 1-h baseline period, average serum cortisol levels did not differ among protocols. However, analysis of individual serum cortisol concentrations at 0830 h, when the baseline period started, revealed that all subjects after the low insulin-ante-hypo had cortisol concentrations in the lower half of the reference range (5 15 g/dl), whereas five subjects in the control group had a cortisol concentration in the upper half of the reference range (15 25 g/dl; P 0.05; Fig. 3). During the stepwise hypoglycemia, serum cortisol levels increased in all protocols (all P ; Fig. 2). This cortisol increase was significantly lower after the low insulin-ante- FIG. 2. Mean (SEM) plasma ACTH concentrations (top) during the subsequent stepwise hypoglycemic clamp and serum cortisol concentrations (bottom) during the antecedent (left) and subsequent stepwise (right) hypoglycemic clamps. Open triangle (dotted lines), Control group; black triangle (thin lines), low insulin-ante-hypo protocol; black square (thick lines), high insulin-ante-hypo protocol.

4 1554 FRUEHWALD-SCHULTES ET AL. JCE&M 1999 Vol 84 No 5 TABLE 1. Mean (SEM) for all parameters during the baseline period; for plasma glucose, serum insulin, and serum C peptide at the end (360 min) of the stepwise hypoglycemic clamp; and for the increase in the counterregulatory hormones and hypoglycemic symptoms Control group Low insulin-ante-hypo protocol High insulin-ante-hypo protocol Plasma glucose (mg/dl) Baseline 92.2 (2.0) 96.4 (2.0) (1.9) a End of the clamp 44.7 (0.6) 45.9 (0.4) 46.5 (0.7) Insulin ( U/mL) Baseline 7.7 (0.6) 10.5 (1.1) b 10.3 (0.7) c End of the clamp (8.3) 92.6 (6.0) 98.1 (4.8) ACTH (pg/ml) Baseline 25.1 (3.5) 15.2 (1.2) b 15.4 (1.9) b Increase during the clamp d 91.8 (10.5) 48.1 (9.4) c 62.1 (13.7) Cortisol ( g/dl) Baseline 10.3 (1.1) 8.9 (0.5) 9.0 (0.6) Increase during the clamp d 13.3 (1.6) 9.6 (1.1) b 13.4 (1.6) e Norepinephrine (pg/ml) Baseline 194 (22) 196 (20) 178 (12) Increase during the clamp d 265 (44) 133 (31) b 201 (23) e Epinephrine (pg/ml) Baseline 30 (4) 31 (5) 27 (4) Increase during the clamp d 972 (160) 624 (91) f 689 (90) GH (ng/ml) Baseline 1.0 (0.05) 1.0 (0.04) 0.9 (0.01) Increase during the clamp d 30.1 (2.0) 19.1 (2.7) a 21.7 (2.3) c Glucagon (pg/ml) Baseline (22.5) (11.1) (13.8) Increase during the clamp d 31.7 (12.5) 12.1 (8.7) 31.1 (8.4) e Autonomic symptom score Baseline 2.73 (0.50) 2.73 (0.57) 2.87 (0.70) Increase during the clamp d 8.53 (0.93) 7.27 (0.79) 8.33 (1.04) Neuroglycopenic symptom score Baseline 4.87 (0.72) 4.47 (0.70) 4.80 (0.86) Increase during the clamp d 3.73 (0.96) 2.13 (0.60) 4.13 (0.86) e a P vs. control group. P 0.05 vs. control group. P 0.01 vs. control group. Value end of the clamp value baseline. e P 0.05 vs. low insulin-ante-hypo protocol. f P 0.1 vs. control group. FIG. 3. Individual cortisol concentrations under basal conditions (left) and after 360 min of hypoglycemia (right). Dashed lines indicate the mean (left;15 g/dl) and the upper limit (right; 25 g/dl) of the reference range. *, P 0.05 (by Fisher s exact test and McNemar s test) for comparison of the number of subjects in each protocol with a basal serum cortisol concentration above the mean (left) and a hypoglycemia-stimulated serum cortisol concentration above the upper limit (right) of the reference range. hypo than in the control group (P 0.05), whereas the effect of the high insulin-ante-hypo remained without significance (P 0.92). The increase in serum cortisol was greater after the high than after the low insulin-ante-hypo (P 0.05). Analysis of individual peak serum cortisol levels at this time revealed that all subjects after the low insulin-ante-hypo had a cortisol concentration within the reference range (5 25 g/ dl), whereas five subjects after the high insulin-ante-hypo and five subjects in the control group had levels above the reference range ( 25 g/ml; both P 0.05; Fig. 3).

5 INSULIN AND HYPOGLYCEMIC COUNTERREGULATION 1555 During stepwise hypoglycemia, plasma norepinephrine and epinephrine levels as well as serum GH levels increased in all protocols (all P 0.001; Fig. 4). Compared to values in the control group, the increases in norepinephrine and epinephrine were blunted after the low insulin-ante-hypo (P 0.05 and P 0.07), but not after the high insulin-ante-hypo (P 0.19 and P 0.13, respectively). The norepinephrine increase was significantly greater after the high than after the low insulin-ante-hypo (P 0.05). The increase in GH was blunted after both the low and high insulin-ante-hypo (P and P 0.01) compared to that in the control group. Serum glucagon levels significantly increased after the high insulin-ante-hypo (P 0.01) and in the control group (P 0.05), but not after the low insulin-ante-hypo (P 0.12; Fig. 4), during stepwise hypoglycemia. The increase in glucagon was significantly greater after the high than after the low insulin-ante-hypo (P 0.05). When data during stepwise hypoglycemic clamps from all protocols were pooled, significant correlations were found between the increases in ACTH and norepinephrine (r 0.36; P 0.05), the increases in cortisol and norepinephrine (r 0.46; P 0.001), the increases in ACTH and epinephrine (r 0.52; P ), and the increases in cortisol and epinephrine (r 0.51; P ). Both autonomic and neuroglycopenic symptom scores increased in all protocols during stepwise hypoglycemia (Fig. 5). The increase in neuroglycopenic symptom score was significantly greater after the high than after the low insulinante-hypo (P 0.05). Discussion The present data demonstrate that a single 2-h episode of even mild hypoglycemia (56 mg/dl) reduces the counterregulatory endocrine response to subsequent hypoglycemia h later. High insulin levels during the antecedent hypoglycemia resulted in higher cortisol, norepinephrine, and glucagon responses to subsequent hypoglycemia than lower insulin levels. Recurrent episodes of hypoglycemia have been demonstrated to reduce subsequent endocrine counterregulation (4 10). Here, we show that a single episode of very mild hypoglycemia (56 mg/dl) causes a reduction of neuroendocrine counterregulation that is still readily discernible about 24 h later. A similar effect of a single hypoglycemic episode has previously been shown in healthy (18) and diabetic (19) humans. The glycemic levels during antecedent hypoglycemia in those studies, however, were lower (46 50 mg/dl) than those in the present study. This difference is noteworthy, because the plasma glucose level during antecedent hypoglycemia has been shown to be a major determinant of the effects on subsequent counterregulation (8). Heller et al. (5) reported a reduced counterregulatory response 18 h after one mild hypoglycemic episode (plasma FIG. 4. Mean (SEM) plasma norepinephrine (top, left), plasma epinephrine (top, right), serum GH (bottom, left), and serum glucagon (bottom, right) concentrations during subsequent stepwise hypoglycemic clamp. Open triangle (dotted lines), Control group; black triangle (thin lines), low insulin-ante-hypo protocol; black square (thick lines), high insulin-ante-hypo protocol.

6 1556 FRUEHWALD-SCHULTES ET AL. JCE&M 1999 Vol 84 No 5 FIG. 5. Mean (SEM) autonomic (top) and neuroglycopenic symptom scores during subsequent stepwise hypoglycemic clamp. Open triangle (dotted lines), Control group; black triangle (thin lines), low insulin-ante-hypo protocol; black square (thick lines), high insulin-antehypo protocol. glucose, 54 mg/dl). The present data extend those findings by showing that counterregulation is still reduced even 24 h after an antecedent hypoglycemia. Together, the prolonged effects of even mild hypoglycemia on subsequent counterregulation underline the importance of scrupulously avoiding even mild hypoglycemic episodes in patients with diabetes. Insulin exerted a moderate, but significant, protective effect on subsequent counterregulation. Responses of cortisol, norepinephrine, and glucagon were higher after high than after low insulin antecedent hypoglycemia. It appears possible that the greater dextrose infusion rates during the high than the low insulin antecedent hypoglycemia may have contributed to the observed preserving effect of insulin on subsequent counterregulation. In summary, the present results suggest that insulin exhibits prolonged effects on several components of hypoglycemic counterregulation that may at least in part overcome the adverse effects of antecedent hypoglycemia on subsequent counterregulation. High insulin levels also acutely enhanced the counterregulatory cortisol response to antecedent hypoglycemia. This finding is in line with the results of several foregoing studies (20 24). Recently, we demonstrated that insulin increases cortisol levels even during euglycemia (25). As insulin receptors have been found at all levels of the hypothalamic-pituitary-adrenal (HPA) axis, i.e. the hypothalamus (26), pituitary (27), and adrenal gland (28), one may speculate that insulin increases HPA activity. Consistent with the present data, decreased basal cortisol levels after antecedent hypoglycemia have also been found in several previous studies (7, 8). From the present data, it appears that the effect of antecedent hypoglycemia was somewhat more consistent on basal ACTH than on cortisol levels. Therefore, it is possible that high cortisol levels during the antecedent hypoglycemia suppressed basal as well as hypoglycemia-induced HPA activity during the following day by a prolonged negative feedback inhibition. Previous research (29) as well as the present observations of distinct correlations between catecholaminergic and ACTH/cortisol responses may indicate a close relationship between activation of the HPA system and the sympatho-adrenal system. Thus, the diminished counterregulatory HPA response, caused by feedback effects of cortisol, may have also contributed to the reduced sympatho-adrenal response after antecedent hypoglycemia. This interpretation of the present data would support the hypothesis of Davis et al. that hypoglycemia-induced increases in plasma cortisol levels represent a major mechanism responsible for subsequent hypoglycemic counterregulatory failure (30). On the other hand, high insulin levels during the antecedent hypoglycemia induced a more distinct rise in cortisol than the low insulin levels, but also preserved subsequent counterregulation. These results seem to argue against the hypothesis by Davis et al. (30). However, it is also possible that the preserving effects of insulin on subsequent counterregulation may have overridden the putative detrimental effects of cortisol. The present study provides the first evidence for a prolonged insulin effect on subsequent counterregulation. It should be pointed out that plasma insulin concentrations during both sets of the antecedent hypoglycemia were supraphysiolgical. Therefore, the clinical relevance and the strength of this insulin effect in a more physiological concentrations cannot directly be determined here. However, in light of findings indicating a sigmoid dose-response relation for the effects of insulin on various other physiological parameters (31), the effects of insulin on hypoglycemic counterregulation presumably will follow a similar dose-response curve. Thus, insulin effects in the physiological range may be even more pronounced than the effects observed here. In summary, the present study demonstrates that a single episode of mild hypoglycemia (56 mg/dl) reduces the counterregulatory response to subsequent hypoglycemia h later. An amplifying effect of insulin on the immediate counterregulatory response to hypoglycemia has been shown in previous studies. Our findings suggest that this amplifying effect may even be extended to a hypoglycemic episode on the next day. Thus, hyperinsulinemia may prevent the development of hypoglycemia-associated counterregulatory failure. Acknowledgments We thank Christiane Zinke and Steffi Baxmann for their expert and invaluable laboratory assistance, and Anja Otterbein for her organiza-

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