Effect of Glycemic Control on Glucose Counterregulation During Hypoglycemia MANDEEP BAJAJ, MD DONALD C. SIMONSON, MD

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1 Pathophysiology/Complications N A L A R T I C L E Effect of Glycemic Control on Glucose Counterregulation During Hypoglycemia CAROL J. LEVY, MD BRENDAN T. KINSLEY, MD, MRCPI MANDEEP BAJAJ, MD DONALD C. SIMONSON, MD OBJECTIVE We examined the effect of glycemic control of NIDDM on counterregulatory hormone responses to hypoglycemia and compared the effect with that seen in patients with IDDM. RESEARCH DESIGN AND METHODS Eleven subjects with NIDDM and eight ageand weight-matched control subjects and ten subjects with IDDM and ten age- and weightmatched control subjects were studied. All subjects underwent a stepped hypoglycemic-hyperinsulinemic clamp study during which plasma glucose levels were lowered in a stepwise manner from 5.0 to 2.2 mmol/1 in steps of 0.6 mmol/1 every 30 min. Counterregulatory hormones (epinephrine, norepinephrine, glucagon, ACTH, cortisol, and growth hormone [GH]) were measured, and a symptom survey was administered during the last 10 min of each 30- min interval. RESULTS The threshold for release of epinephrine, norepinephrine, ACTH, and cortisol occurred at higher plasma glucose levels in NIDDM than in IDDM patients (P < ). The glucose threshold for release of epinephrine and norepinephrine correlated with glycemic control as measured by glycosylated hemoglobin (P < ). However, for a given level of glycemic control, the threshold for release of epinephrine and norepinephrine occurred at a higher glucose level in NIDDM versus IDDM patients (P < ). At the nadir level of hypoglycemia, glucagon, ACTH, and cortisol levels were all higher in NIDDM compared with IDDM subjects, whereas GH levels were lower. CONCLUSIONS Glycemic control alters counterregulatory responses to hypoglycemia in NIDDM as has been previously reported in IDDM. However, at similar levels of glycemic control, NIDDM patients release counterregulatory hormones at a higher plasma glucose level than patients with IDDM. In addition, subjects with NIDDM maintain their glucagon response to hypoglycemia. These data suggest that patients with NIDDM may be at reduced risk of severe hypoglycemia when compared with a group of IDDM patients in similar glycemic control, thus providing a more favorable risk-benefit ratio for intensive diabetes therapy in NIDDM. The Diabetes Control and Complications Trial (DCCT) demonstrated that intensive insulin therapy in subjects with IDDM decreased the incidence and progression of the microvascular complications of diabetes. However, this study and others have demonstrated that improvement in glycemic control is achieved at the expense of an increased frequency and severity of hypoglycemia (1-4). Based on the results of the DCCT, intensive diabetes management also has been recommended for subjects with NIDDM (5,6), despite the fact that the counterregulatory responses to hypoglycemia and the relationship between glycemic control and glucose counterregulation in NIDDM remain uncertain (7-13). A number of studies have found that NIDDM subjects experience less hypoglycemia than IDDM subjects during intensive From thejoslin Diabetes Center (C.J.L., B.T.K., M.B., D.C.S.), and the Brigham and Women's Hospital (B.T.K., D.C.S.), Harvard Medical School, Boston, Massachusetts. Address correspondence and reprint requests to Carol J. Levy, MD, Division of Endocrinology, Diabetes and Metabolism, Mount Sinai Medical Center, 1 Gustave L. Levy Place, Box 1055, New York, NY Received for publication 4 December 1997 and accepted in revised form 21 April Abbreviations: DCCT, Diabetes Control and Complications Trial; GH, growth hormone. therapy (14). For example, the results of the recent Veterans Administration (VA) feasibility trial of intensive diabetes therapy in insulin-treated NIDDM subjects reported a rate of severe hypoglycemia at least 20 times less than the rate reported in the intensively treated group in the DCCT (15,16). Data from the U.K. Prospective Diabetes Study (UKPDS) trial also revealed lower rates of hypoglycemia than the DCCT (13). We therefore decided to study non-insulintreated NIDDM subjects across a wide range of glycemic control to determine whether 1) counterregulatory responses and symptoms during hypoglycemia differ in NIDDM patients and age-matched control subjects, 2) if hormone responses to hypoglycemia differ between NIDDM and IDDM subjects, and 3) if glycemic control affects counterregulation in NIDDM. RESEARCH DESIGN AND METHODS Study participants In this study, 39 subjects were examined (Table 1). Eleven subjects with NIDDM were compared with eight control subjects of comparable age and BMI (older control group). Both groups were moderately obese. The NIDDM subjects had a wide range of glycemic control (HbA l %; normal range, %). Among the NIDDM group, seven subjects were taking sulfonylureas for diabetes management, and four were treated with diet. Ten subjects with IDDM and ten agematched control subjects (younger control group) were used for comparison. These subjects were significantly younger and thinner (P < 0.001) than the other two groups. The IDDM group was matched for disease duration and degree of glycemic control with the NIDDM group. Some of the insulin clamp data from the IDDM group have been previously reported (17). Diabetic subjects were in good health without clinically significant vascular complications or autonomic neuropathy as measured by heart rate variation during slow deep breathing at a rate of six breaths/minute and in response to the Val DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST 1998

2 Levy and Associates Table 1 Demographic characteristics of the study subjects N1DDM patients Older control subjects IDDM patients Younger control subjects n Age (years) Sex (M/F) BM1 (kg/m 2 ) Diabetes duration (years) HbA t (%) (range) ±7* 9/ ±3.3* 8±5 9.7 ± 2.3T ( ) 8 53 ±3* 3/5 29 ±1* 6.7 ± 0.2 ( ) ±6 5/ ±2.6 9± ± 3.2t ( ) Data are means ± SD. *P < vs. IDDM and younger control groups; TP < 0.05 vs. age-matched control groups ±1 5/5 22 ±1 6.0 ±0.1 ( ) salva maneuver (17). The older control group was screened for impaired glucose tolerance by a standard 2-h oral glucose tolerance test, and all had normal glucose tolerance. None of the participants were taking any medications known to interfere with glucose metabolism or hormone responses to hypoglycemia. Subjects were informed of the possible risks of the study and gave voluntary written informed consent before the study. The protocol was approved by the Joslin Diabetes Center Committee on Human Studies. Experimental procedures All studies started at 8:00 A.M. after a 10- to 12-h overnight fast. Study subjects were admitted to the Clinical Research Center at 7:30 A.M. on the day of the study. NIDDM subjects withheld their oral agents for 48 h before being studied; IDDM subjects received their last subcutaneous insulin injection either before dinner or at 10:00 P.M., based on their insulin regimen, and did not take their morning insulin dose on the day of the study. To decrease the risk of unrecognized nocturnal hypoglycemia, patients were instructed to eat a small snack of carbohydrate and protein if their bedtime blood glucose was <5.6 mmol/1. Studies were postponed at least 1 week if subjects experienced symptomatic hypoglycemia or a blood glucose <3.3 mmol/1 during the previous 24 h. On the morning of the study, a catheter was inserted into an antecubital vein of the nondominant hand for administration of test substances, and a second catheter was placed retrogradely into a vein on the dorsum or wrist for blood sampling. The hand was placed in a heated box (70 C) to ensure arterialization of venous blood (19). In the diabetic subjects, the plasma glucose was initially stabilized between 5.0 and 9.0 mmol/1 for 1 h with a low-dose insulin infusion at pmol kg" 1 min" 1 ( mu kg" 1 min" 1 ). Thereafter, the glucose levels were permitted to slowly decline to a target level of 5.0 mmol/1 by the beginning of the clamp study Three baseline blood samples were taken, and a symptom survey was administered during thefinal30 min before starting the glucose clamp. A primed continuous infusion of 12 pmol kg" 1 min" 1 (2 mu kg" 1 min" 1 ) of regular human insulin (Eli Lilly, Indianapolis, IN) was begun and continued for 180 min. Six of the NIDDM subjects needed increased insulin infusions during the latter part of the study to achieve appropriate levels of hypoglycemia, 18 pmol kg" 1 min" 1 (3 mu kg" 1 min" 1 ) in two subjects, 24 pmol kg" 1 min" 1 (4 mu kg" 1 min" 1 ) in three subjects, and 36 pmol kg" 1 min" 1 (6 mu kg" 1 min" 1 ) in one subject. Plasma glucose levels were measured at 5-min intervals, and the glucose clamp technique (20,21) was used to produce a stepwise decline in plasma glucose from 5.0 to 4.4, 3.9, 3.3, 2.8, and 2.2 mmol/1 (i.e., 90, 80, 70, 60, 50, and 40 mg/dl, respectively) at 30-min intervals. During the last 10 min of each 30-min interval, plasma samples were obtained for measurement of epinephrine, norepinephrine, glucagon, ACTH, cortisol, and GH, and a symptom survey was administered. Subjects were blinded to their plasma glucose concentration throughout the study. The symptom survey consisted of a selfadministered checklist for the intensity of the following 10 symptoms: confusion, difficulty in thinking, faintness, dizziness, blurry vision, shakiness, pounding of the heart, nervousness, sweating, and feeling different in any way Subjects rated the intensity of each symptom from 0 (none) to 10 (severe). The sum of the scores for the first five items were used to determine the "neuroglycopenic symptom score," and the sum of the scores for the next four items were used to determine the "autonomic symptom score." Detailed assessment of the symptoms of hypoglycemia using principal components (factor) analysis by Hepburn et al. (22,23) and Deary et al. (24) indicate that the symptoms used in the questionnaire are correctly classified into the autonomic and neuroglycopenic symptom groups, although not all of the most common symptoms associated with hypoglycemia are used. Analyses Plasma glucose was measured at the bedside using the glucose oxidase method (YSI, Yellow Springs, OH). Plasma insulin was determined by a double-antibody radioimmunoassay (25). In the diabetic subjects, free insulin assays were performed after treating the plasma with polyethylene glycol to precipitate the antibody-bound insulin. Total glycosylated hemoglobin was measured by agar gel electrophoresis with the GLYTRAC glycosylated hemoglobin set (Corning Medical, Palo Alto, CA) after removal of the labile component (26). Plasma epinephrine and norepinephrine levels were determined by radioenzymatic assay (27). Growth hormone (GH) (28), glucagon (29), cortisol (30), and adrenocorticotropin (Nichols Laboratories, San Juan Capistrano, CA) (31) levels were determined using standard radioimmunoassay procedures. Data are presented as means ± SEM except for the demographic data in Table 1, which are presented as means ± SD. Comparisons between groups were assessed by using Student's t test for paired and unpaired data as appropriate or by using analysis of variance (ANOVA) with repeated measures where appropriate. For data that were not normally distributed, comparisons between groups were made using the Mann-Whitney 17 and Kruskal- Wallis tests. Correlation coefficients were determined by linear regression. Multiple regression analysis was used to examine the effect of multiple independent variables (e.g., age, duration of diabetes, BMI, HbA x ) on a single dependent variable (e.g., the DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST

3 Hypoglycemia and counterregulation in NIDDM mean basal for each diabetic or control group (35). RESULTS Basal Time (minutes) Figure 1 Plasma glucose levels during hypoglycemic clamp studies in 11 subjects with NIDDM ( ), 8 age- and weight-matched older control subjects ( ), JO subjects with IDDM (A), and 10 age- and weight-matched younger control subjects (O). *P < 0.05 NIDDM vs. IDDM; TP < 0.0J IDDM vs. young control subjects. counterregulatory response to hypoglycemia). All statistical analyses were performed using the SYSTAT statistical software program (Evanston, IL). The glucose threshold required for the stimulation of release of each counterregulatory hormone was determined as the plasma glucose at which the hormone achieved a sustained increment above basal as previously described for IDDM subjects (32). This predefined increment was defined as a rise over baseline that occurred in at least two consecutive samples and was 410 pmol/1 for epinephrine, 0.3 nmol/1 for norepinephrine, 190 nmol/1 for cortisol, and 7 ug/1 for GH. The validity of these comparisons has previously been described in detail (32-34). Increments for ACTH and glucagon were defined in statistical terms as two standard deviations above the Glucose and insulin Glucose levels were 5.7 ± 0.6 mmol/1 in the NIDDM group and 4.6 ± 0.3 mmol/1 in the older control group at the start of the insulin clamp protocol (P = NS). Glucose levels were higher in the NIDDM group compared with the older control group at the 30-min time point but did not differ for the remainder of the study (Fig. 1). Basal glucose levels were higher in the IDDM group compared with the younger control group (6.4 ± 0.4 vs. 5.1 ± 0.1 mmol/1, P < 0.01), but did not differ for the remainder of the study (Fig. 1). Mean insulin levels during the study were higher in the NIDDM group (5,142 ± 1,080 pmol/1) compared with the older control group (1,422 ± 66 pmol/1, P < 0.01), the IDDM group (792 ± 72 pmol/1, P < 0.001), and the younger control group (940 ± 97 pmol/1, P < 0.01). Mean insulin levels were also higher in the older control group when compared with both the IDDM and younger control groups (P < 0.001). Counterregulatory hormones Glucagon. Basal glucagon levels were higher in the NIDDM than IDDM subjects (128 ± 14 vs. 79 ± 19 ng/1, P < 0.05). 300 B Q. <D C Q. LU 2000 Basal Target Glucose (mmol/1) Basal Target Glucose (mmol/1) Figure 2 A: Plasma glucagon levels during hypoglycemic clamp studies in 11 subjects with NIDDM ( ), 8 age- and weight-matched older control subjects ( ), 10 subjects with IDDM (A), and 10 age- and weight-matched younger control subjects (O). *P < 0.05 NIDDM vs. IDDM, TP < 0.05 older controls vs. younger controls. B: Plasma epinephrine levels during hypoglycemic clamp studies in 11 subjects with NIDDM ( ), 8 age- and weight-matched older control subject subjects ( ), 10 subjects with IDDM (A), and 10 age- and weight-matched younger control subjects (O). *P < 0.01 NIDDM vs. IDDM DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST 1998

4 Levy and Associates Table 2 Glucose thresholds for release of counterregulatory hormones during hypoglycemia NIDDM Older control subjects IDDM Younger control subjects Epinephrine 4.1 ±0.2t 3.0 ± ±0.3 ±0.2 Norepinephrine 4.0 ± 0.3t 3.4 db ± ±0.2 ACTH 3. 7 ±0.2* 3.0 ± ± ±0.4 Cortisol 3.5 ± 0.2*1= 2.6 ± ± ±0.1 GH 3.4 ±0. 2* 2. 6± l± ±0.2 Data are means ± SEM, expressed in millimoles per liter. Statistical comparisons reported only for NIDDM vs. older control subjects, IDDM versus younger control subjects, NIDDM vs. IDDM, and older control vs. younger control subjects. *P < 0.05 versus older control group; tp < 0.05 vs. IDDM group; TP < 0.01 vs. IDDM group. Glucagon levels remained significantly higher in the NIDDM subjects at all glucose levels from 3.9 to 2.8 mmol/1 (P < 0.05; Fig. 2A). Glucagon levels did not differ between NIDDM subjects and the matched older control group. Similarly, glucagon levels did not differ between the older and younger control groups, except at a glucose of 2.2 mmol/1 when the younger group was significantly higher (277 ± 32 vs. 156 ± 42 ng/1, P < 0.05; Fig. 1A). Glucose thresholds for glucagon release are not reported because of wide standard deviations in mean basal values for the groups. However, the incremental glucagon response was significantly greater in the NIDDM group compared with the older control group at a glucose level of 3.9 mmol/1 (22 ± 15 vs. -24 ± 12 ng/1, P < 0.05), suggesting that the glucose threshold for glucagon release may be altered in the NIDDM group. Epinephrine. Basal levels of epinephrine did not differ among the four groups (NIDDM = 257 ± 49, older control subjects = 337 ± 83, IDDM = 285 ± 48, younger control subjects = 223 ± 56 pmol/1). Epinephrine levels, although higher in the NIDDM group, did not differ statistically from those in the IDDM group during the clamp study (Fig. 2B). The epinephrine response in the NIDDM group was greater than that of the older control group at glucose levels of 3.9 mmol/1 (1,894 ± 383 vs. 470 ± 224 pmol/1, P < 0.01) and 3.3 mmol/1 (3,653 ± 759 vs. 770 ± 221 pmol/1, P < 0.01). Epinephrine levels did not differ between older and younger control subjects during the study (Fig. IB). Glucose thresholds for release of epinephrine occurred at a higher plasma glucose level in the NIDDM subjects when compared with the IDDM group (4.1 ± 0.2 vs. 3.1 ± 0.3 mmol/1, P < 0.01; Table 2). In both NIDDM and IDDM, positive correlations existed between HbAj and the glucose level required for epinephrine secretion (r = 0.82, P < 0.01 for NIDDM; r = 0.63, P < 0.05 for IDDM; Fig. 3A). However, at any level of HbA t the glucose threshold in NIDDM subjects for epinephrine release in response to hypoglycemia occurred at a higher glucose level than in IDDM subjects (P < 0.05, Fig. 3A). Norepinephrine. Basal levels of norepinephrine were significantly higher in the NIDDM subjects compared with IDDM subjects (1.51 ± 0.19 vs ± 0.09 nmol/1, P < 0.05; Table 3). Norepinephrine levels remained higher in the NIDDM group compared with the IDDM group at all glucose levels from 5.0 to 2.2 mmol/1 (P < ). Basal norepinephrine levels were also significantly higher in the older control group compared with the younger control group (1.68 ± 0.19 vs ± 0.07 nmol/l, P < 0.01, Table 3), and levels remained higher in the older control group at all glucose levels from 5.0 to 2.8 mmol/1 (P < ). Glucose thresholds for release of norepinephrine occurred at a higher plasma glucose level in the NIDDM subjects when compared with the IDDM group (4.0 ± 0.3 vs. 2.7 ± 0.2 mmol/1, P < 0.01; Table 2). In both NIDDM and IDDM subjects, there was a correlation between HbA t and the glucose level required for norepinephrine secretion (r = 0.74, P < 0.01 for NIDDM; r = 0.77, P < 0.01 for IDDM, Fig. 6 r B HbA1 (%) Figure 3 A: Relationship between glucose threshold (millimols per liter) for epinephrine and HbAi in NIDDM ( ) and IDDM (A) subjects: r = 0.82, P < 0.01 for NIDDM group; r = 0.63, P < 0.05 for IDDM group; P < 0.05 between groups. B: Relationship between glucose threshold (millimoles per liter) for norepinephrine and HbAj in NIDDM ( ) and IDDM (A) subjects: r = 0.74, P < 0.01 for NIDDM group; r = 0.77, P < 0.01 for IDDM group; P < 0.01 between groups. DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST

5 Hypoglycemia and counterregulation in NIDDM Table 3 Basal and nadir (2.2 mmol/l) values for ACTH, cortisol, GH, and norepinephrine during hypoglycemia Norepinephrine (nmol/1) Basal Nadir ACTH (pmol/l) Basal Nadir Cortisol (nmol/1) Basal Nadir GH (pg/1) Basal Nadir Older NIDDM control subjects IDDM 1.51 ± 0.191= 4.4 ± 0.6T 5.3: b : b6.6t 321 ±28 723: t 55=f 1.9 ± 0.3* 13 ± :5t* 1.68 ± ± ± ± ± ±72 5.S)±3 39 ± ± ± ± ± ± ± ± ±8 Younger control subjects ±0.1 ± ± ± ± ± ± ±12 Data are means ± SEM. *P < 0.05 vs. older control group; tp < 0.01 vs. IDDM group; JP < 0.05 vs. IDDM group; P < 0.01 vs. younger control group. 3B). Thus, at any level of HbA b NIDDM subjects began secreting norepinephrine at higher glucose levels than IDDM subjects (P < 0.01, Fig. 3B). ACTH. Basal ACTH levels were not significantly different among the four study groups (Table 3). ACTH levels were significantly higher in NIDDM subjects compared with the IDDM group at all glucose levels from 3.9 to 2.2 mmol/l (P < 0.01, Table 3). The threshold at which release of ACTH occurred was significantly higher in the NIDDM subjects (3.7 ± 0.2 mmol/l) when compared with the older control group (3.0 ± 0.2 mmol/l, P < 0.05, Table 2). Glucose thresholds for ACTH release were slightly, but not significantly, higher in NIDDM versus IDDM patients (Table 2). Cortisol. Basal cortisol levels did not differ between study groups (Table 3). Cortisol levels were greater in the NIDDM than IDDM group at glucose levels of 3.9, 3.3, 2.8, and 2.2 mmol/l (P < 0.05, Table 3). Cortisol levels were significantly higher in the NIDDM group compared with the older control group at glucose levels of 3.3 and 2.8 mmol/l (P < 0.01). The glucose threshold for cortisol release occurred at a higher glucose level in the NIDDM group (3.5 ± 0.2 mmol/l) when compared with both the IDDM (2.7 ± 0.3 mmol/l, P < 0.05) and the older control group (2.6 ± 0.2 mmol/l, P < 0.05, Table 2). Growth hormone. Basal GH levels were significantly lower in the NIDDM group when compared with IDDM subjects (1.9 ± 0.3 vs ± 3.6 pg/1, P < 0.05; Table 3). GH levels at the nadir glucose of 2.2 mmol/l were significantly lower in the NIDDM group compared with both the IDDM (P < 0.01) and older control groups (P < 0.05; Table 3). BMI was inversely correlated with GH response during hypoglycemia in the NIDDM group (r = -0.66, P < 0.05) and in the combined NIDDM and older control group (r = -0.54; P < 0.01). GH was secreted at a higher glucose level in the NIDDM group when compared with the age-matched control group (3.4 ± 0.2 vs. 2.6 ± 0.1 mmol/l, P < 0.05; Table 2). Symptoms of hypoglycemia Basal symptom scores for total, autonomic, and neuroglycopenic symptoms did not differ among groups. Although both autonomic and neuroglycopenic symptom scores in both NIDDM and IDDM groups tended to increase at glucose levels above those seen in the control groups, this difference was not statistically significant. At nadir hypoglycemia, symptom scores for total, autonomic, and neuroglycopenic symptoms did not differ among study groups. CONCLUSIONS In the current study, we compared the counterregulatory hormone and symptomatic responses to hypoglycemia in noninsulin-treated NIDDM subjects with IDDM subjects and age- and weight-matched control groups. We determined that there were substantial differences between the diabetic groups in the effect of glycemic control on counterregulation. In the NIDDM subjects J) the glucagon response to hypoglycemia is preserved, 2) the release of epinephrine, norepinephrine, ACTH, and cortisol is greater than in IDDM subjects and age-matched control subjects, and 3) the glucose threshold required for epinephrine and norepinephrine release is significantly higher than in IDDM subjects with similar levels of glycohemoglobin. Thus, for a given level of glycemic control, the release of epinephrine and norepinephrine occurred at a higher glucose level in NIDDM than IDDM patients. Viewed in another way, for a given degree of hypoglycemia, subjects with NIDDM had a more robust counterregulatory hormone response (with the exception of GH) when compared with a group of IDDM subjects in similar glycemic control. Glucagon is the primary counterregulatory hormone against acute hypoglycemia in healthy subjects (36). Subjects with IDDM have a marked impairment in the glucagon response to hypoglycemia and, thus, are dependent on epinephrine for counterregulation during acute hypoglycemia (36,37). This lack of a glucagon response is specific for hypoglycemia alone, and glucagon responses to nonhypoglycemic stimuli remain intact (37,38). The mechanism for this defect is not known, but may be related to alterations in intra-islet cellular communication as a result of deficient insulin secretion (39). In the current study, the glucagon response to hypoglycemia did not differ between NIDDM and the older control group although, as expected, it was higher than in the IDDM group throughout the study (P < 0.05). The incremental glucagon response did not differ between NIDDM and older control subjects except at the 3.9 mmol/l glucose level where it was higher in the NIDDM group, suggesting a possible alteration in the threshold for glucagon secretion. The literature is inconclusive as to whether the glucagon response to hypoglycemia is abnormal in subjects with NIDDM. An elegant study by Shamoon et al. (12) reported a blunted glucagon response to hypoglycemia at 2.8 mmol/l in nine moderately obese NIDDM subjects compared with matched control subjects, confirming a similarfindingby Bolli et al. (10). In contrast, a normal glucagon response to hypoglycemia in NIDDM has been reported in other studies (7-9). In the absence of glucagon, subjects with IDDM are dependent on epinephrine for counterregulation during acute hypoglycemia. However, defects in the epinephrine response to hypoglycemia are known to occur in subjects with well-controlled IDDM or during intensive diabetes therapy caused 1334 DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST 1998

6 Levy and Associates by an increased frequency of hypoglycemia (40-44). In contrast, the preservation of the glucagon response in the NIDDM group in this study may maintain the normal hierarchy of counterregulation and attenuate the hypoglycemia-induced reduction in epinephrine responses. The epinephrine responses to hypoglycemia in the NIDDM group would support this hypothesis. Epinephrine levels in response to graded hypoglycemia were higher in NIDDM subjects compared with a matched control group in this study, and the threshold for release of epinephrine occurred at a higher glucose level in NIDDM subjects when compared with both IDDM and older control subjects (Fig. 3A and Table 2). Previous studies on epinephrine responses to hypoglycemia reported no differences between NIDDM subjects compared with age- and weight-matched control subjects (7-10). However, more recent studies using the insulin clamp technique have suggested that the epinephrine response in NIDDM may be increased and begin at a higher glucose level than in control subjects (11,12). Our study expands on these previous observations by demonstrating that the threshold for epinephrine release occurs at a significantly higher plasma glucose level in NIDDM than IDDM subjects for any given level of glycohemoglobin. Numerous studies in IDDM suggest that this adaptation in glucose threshold for epinephrine release results from the relative frequency of exposure to hypoglycemia (45-47). The normal hierarchy of counterregulatory responses in the NIDDM subjects described above would act to reduce the exposure to hypoglycemia in this patient group and explain the differences in the glucose thresholds between the diabetic groups. Basal levels of norepinephrine were significantly higher in the NIDDM subjects compared with IDDM subjects (1.51 ± 0.19 vs ± 0.09 nmol/1, P < 0.05; Table 3). During hypoglycemia, both the NIDDM and the older control groups had significantly higher norepinephrine levels compared with both the IDDM and younger control groups (Table 3). Previous studies have shown that norepinephrine levels increase with age, caused by both decreased norepinephrine clearance and increased spillover to plasma (48,49). A major effect of hyperinsulinemia to modulate the norepinephrine response to hypoglycemia appears unlikely, because the response to hypoglycemia did not differ between the NIDDM group and the older control group despite differing insulin levels. Using criteria similar to those used for IDDM subjects for calculation of the glucose threshold (32), the threshold for release of norepinephrine occurred at a higher plasma glucose level than for the IDDM group. Moreover, the glucose threshold for norepinephrine correlated with glycemic control as measured by HbAj level (r = 0.74, P < 0.01, Fig. 3B) and occurred at a higher glucose level for a given HbAj level. ACTH responses to hypoglycemia are increased in NIDDM when compared with age-matched control subjects and subjects with IDDM. The glucose threshold for ACTH release occurred at a higher plasma glucose level in the NIDDM group than in the matched control group, but did not differ significantly from that in the IDDM group. Few published data exist on ACTH responses to hypoglycemia in NIDDM subjects. Some studies have reported increased ACTH levels in NIDDM (50,51), while others comparing basal ACTH levels in NIDDM and IDDM subjects have not found a difference (52,53). Data from IDDM subjects suggest that basal ACTH levels may be unchanged (54) or increased (55) compared with matched control groups, while strict glycemic control of IDDM (35) or recurrent exposure to hypoglycemia (56) decreases the ACTH response to hypoglycemia. Cortisol levels paralleled those of ACTH, with higher levels seen in the NIDDM subjects. Ourfindingsare similar to those of Meneilly et al. (11) who studied 10 nonobese noninsulin-treated NIDDM subjects and reported higher cortisol levels in the NIDDM group. Thus, based on the ACTH and cortisol data from this study, it appears that the pituitaryadrenal response to hypoglycemia is certainly not impaired, and may be increased, in subjects with NIDDM. The NIDDM and older control groups tended to have lower GH levels than did the younger study groups. GH responses to hypoglycemia are known to decrease with age and the presence of obesity (57-59). However, our data would suggest that GH responses are further reduced in the NIDDM group when compared with the older control group. Previous studies that compared NIDDM subjects with nondiabetic control subjects have reported either no difference in GH responses (7-9, 12) or reduced GH responses in the NIDDM subjects (10,11). In this study, mean insulin levels were higher in the NIDDM group compared with the other three study groups because six of the eleven NIDDM patients required an increased insulin infusion rate to achieve adequate hypoglycemia. Mean insulin levels in the older control group were also significantly higher than those in the IDDM subjects and younger control group. NIDDM is known to be an insulin-resistant state, independent of the effect of obesity (60), and thus these differences in insulin levels are to be expected. However, there remains the possibility that the higher insulin levels in the NIDDM group may have affected the counterregulatory responses. Data from studies in type 1 diabetes are conflicting on an effect of varying insulin levels on counterregulation, with some studies showing augmentation of epinephrine response (61,62), while others showed either no effect (63,64) or a decrease (65). Previous studies of subjects with type 2 diabetes have not shown a consistent effect of hyperinsulinemia on counterregulation (11,12). The findings of augmented epinephrine with lower insulin levels by both Shamoon et al. (12) and Menielly et al. (11), and lower epinephrine levels in our subjects, would make it unlikely that hyperinsulinemia increased our NIDDM subjects' responses. In addition, none of these studies showed increased glucagon responses with higher insulin levels. The design of our current study, which sought to match the glucose level between groups, does not allow resolution of this issue. Further studies of hypoglycemia with varying levels of hyperinsulinemia in welldefined groups of subjects with type 2 diabetes will be needed to clarify this issue. We did not detect differences in symptom scores between the study groups. Basal symptoms for total, neurogenic, and neuroglycopenic symptoms did not differ among groups. The total symptom score at baseline was 0.5 ± 0.3 versus 0.4 ± 0.4 in type 2 subjects versus the older control group. Although both neurogenic and neuroglycopenic scores in both diabetic groups tended to increase at glucose levels above those seen in control groups, this difference was not statistically significant. At nadir hypoglycemia, symptoms for total, neurogenic, and neuroglycopenic symptoms did not differ among study groups. The total symptom score was 26 ± 7 versus 29 ± 16 in the NIDDM compared with older control group at 2.2 mmol/1. Similar data have been reported by Hepburn et al. (66) who compared hypoglycemic symptom experiences in insulin-treated NIDDM and IDDM subjects and reported that the range and prevalence of specific symptoms were similar to DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST

7 Hypoglycemia and counterregulation in NIDDM those described by the patients with IDDM. Meneilly et al. (11), using a symptom survey similar to that used in this study, also detected no difference in symptom scores in 10 nonobese elderly subjects with NIDDM when compared with a matched control group. Recently, Amiel et al. (13) reported that improving glycemic control with insulin in subjects with poorly controlled type 2 diabetes lowered the glucose level at which there is a deterioration in cognitive function. None of the subjects in our study were insulin treated, and therefore we would not expect that they would have experienced the expected increase in hypoglycemia frequency associated with insulin therapy. Therefore, based on our data, we can conclude only that symptom perception of hypoglycemia is not impaired in noninsulintreated subjects with type 2 diabetes. Thus, although in our study it appears that the perception of hypoglycemia is not impaired in subjects with NIDDM, this area deserves further evaluation. In summary, this study suggests that the preservation of the glucagon response may protect NIDDM patients from the frequent episodes of hypoglycemia that are typically observed in IDDM. Because of the diminished frequency of hypoglycemia, the downregulation of the glucose threshold for release of counterregulatoty hormones, particularly epinephrine, is lessened. Thus, with intact glucagon and epinephrine responses, NIDDM subjects retain a relatively normal hierarchy of counterregulation to hypoglycemia and are likely to be at lower risk for severe hypoglycemia than IDDM patients despite similar levels of glycemic control. This relative protection from hypoglycemia in NIDDM may enable clinicians to optimize glycemic control in these subjects with less risk of severe hypoglycemia. Clearly, further evaluation of hormonal and symptom responses during hypoglycemia in NIDDM subjects, especially when intensively treated with insulin, will need to be performed to further evaluate these issues. Acknowledgments This work is supported in part by a fellowship grant from the Juvenile Diabetes Foundation International (B.T.K.), a Career Development Award from the American Diabetes Association (B.T.K.), a grant from the Adler Foundation (D.C.S), and National Institutes of Health Grant DK (Diabetes and Endocrinology Research Center at the Joslin Diabetes Center) and RR (General Clinical Research Center at Brigham and Women's Hospital). This study was presented in part at the 55th meeting of the American Diabetes Association, Atlanta GA, June, We thank Julia McClure for expert assistance with the clinical protocols and Irene Reske and Marta Grinbergs for careful performance of the laboratory assays. References 1. 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9 Hypoglycemia and counterregulation in NIDDM betologia 37: ,1994 terregulatory response to hypoglycemia by Scougal IJ, Frier BM: Frequency and symp- 65. Diamond MP, Hallarman L, Starick-Zych K, insulin per se. J Clin Endocrinol Metab toms of hypoglycemia experienced by Jones TW, Connolly-Howard M, Tambor- 72: ,1991 patients with type 2 diabetes treated with lane WySherwinRS: Suppression of coun- 66. Hepburn DA, McLeod KM, Pell ACH, insulin. Diabet Med 10: , DIABETES CARE, VOLUME 21, NUMBER 8, AUGUST 1998