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Glycemic Thresholds for Activation of Counterregulatory Hormone and Symptom Responses in Islet Transplant Recipients

Department of Medicine, Divisions of Endocrinology, Diabetes, and Metabolism (M.R.R., M.H.S., R.M., K.L.T.) and Nephrology (S.K.), Department of Surgery, Division of Transplantation (J.F.M., A.N.), and the Monell Chemical Senses Center (K.L.T.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6149

Address all correspondence and requests for reprints to: Michael R. Rickels, M.D., University of Pennsylvania School of Medicine, Division of Endocrinology, Diabetes, and Metabolism, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149. E-mail: rickels{at}mail.med.upenn.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: In patients with type 1 diabetes and reduced awareness of hypoglycemia, the glycemic thresholds for activation of counterregulatory hormone and symptom responses to hypoglycemia are impaired, in part due to recurrent episodes of hypoglycemia. Islet transplantation can ameliorate occurrences of hypoglycemia in these patients.

Objective: The objective of the study was to determine whether the avoidance of hypoglycemia achieved through islet transplantation results in improved glycemic thresholds for counterregulatory responses.

Setting: The study was conducted at a general clinical research center.

Participants: Seven islet transplant recipients, six type 1 diabetic, and eight nondiabetic control subjects participated in the study.

Intervention: We performed a stepped hyperinsulinemic hypoglycemic clamp and, in 12 subjects, a paired hyperinsulinemic euglycemic clamp to calculate the glycemic thresholds for and magnitude of counterregulatory responses.

Results: The glycemic thresholds for all counterregulatory hormone and symptom responses in the islet transplant group were comparable with normal and higher than in the type 1 diabetes group (P < 0.01 for glucagon; P < 0.05 for epinephrine). The magnitude of the glucagon and epinephrine responses in the islet transplant group, although greater than in the type 1 diabetes group (P < 0.05 for both), remained less than normal (P < 0.01 for glucagon; P < 0.05 for epinephrine). The magnitude of GH secretion in the islet transplant group was comparable with normal and greater than in the type 1 diabetes group (P < 0.05).

Conclusions: The glycemic thresholds for activation of counterregulatory hormone and symptom responses appear normal after islet transplantation; however, the magnitudes of the glucagon and epinephrine responses remain impaired.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IN ESTABLISHED TYPE 1 diabetes, defective glucose counterregulation exists because of an absolute deficiency in endogenous insulin secretion and a related defect in glucagon secretion in response to hypoglycemia (1). Normally, inhibition of endogenous insulin secretion and activation of glucagon secretion are sufficient to increase hepatic glucose production and prevent hypoglycemia; however, in the absence of these responses in type 1 diabetes, additional sympathoadrenal (epinephrine secretion and autonomic symptom generation) and pituitary-adrenal (GH and cortisol secretion) counterregulatory responses to hypoglycemia become necessary to increase hepatic glucose production, decrease peripheral glucose use, and promote food ingestion, which together may correct low blood glucose (1). Unfortunately, both the magnitude and glycemic threshold (i.e. the glucose level that elicits the response) of these hormonal and symptom responses to hypoglycemia are impaired in long-standing type 1 diabetes and result in the syndrome of reduced hypoglycemia awareness and its substantially increased risk of life-threatening hypoglycemic episodes (2). The shifting of glycemic thresholds for counterregulatory responses to lower plasma glucose concentrations is best explained by the hypothesis of hypoglycemia-associated autonomic failure (HAAF), which posits recurrent episodes of hypoglycemia blunt subsequent sympathoadrenal and pituitary-adrenal responses to hypoglycemia (3). Such recurrent episodes are often asymptomatic and can occur with sleep and exercise (4). Strict avoidance of hypoglycemia in subjects with type 1 diabetes can normalize the glycemic thresholds for counterregulatory epinephrine, autonomic symptom, and GH responses and consequently reestablish awareness of hypoglycemia (5, 6, 7, 8, 9).

Islet transplantation is being investigated in the United States and used in other countries as an approach to ameliorating severe hypoglycemic episodes in patients with type 1 diabetes and the syndrome of reduced awareness of hypoglycemia (10). When an islet transplant successfully engrafts, as determined by the restoration of endogenous insulin secretion (measured most often as the serum C-peptide concentration), the development of severe hypoglycemic episodes is eliminated, an effect than can last for up to 5 yr (11). Because the majority of islet transplant recipients require some insulin therapy, albeit at a reduced dose, by the second year after transplant, the clinical finding of hypoglycemic avoidance suggests a possible improvement in glucose counterregulatory mechanisms. Studies using continuous glucose monitoring systems in islet transplant recipients have demonstrated significant decreases (in insulin requiring subjects) to abolition (in insulin independent subjects) of time spent in the hypoglycemic range [<60 mg/dl (3.3 mmol/liter)] (12, 13, 14). Because even moderate hypoglycemia between 50 and 58 mg/dl (2.8–3.2 mmol/liter) has been shown experimentally to impair subsequent counterregulatory responses to hypoglycemia (15), the avoidance of hypoglycemia after islet transplantation would be expected to ameliorate HAAF and improve the glycemic thresholds for activation of counterregulatory responses. We previously demonstrated that intrahepatic transplanted islets in type 1 diabetic patients respond appropriately to hyperinsulinemic hypoglycemia by suppressing endogenous insulin secretion and partially restoring counterregulatory glucagon secretion (16). In the present communication, we report the glycemic thresholds for activation of counterregulatory hormone secretion and symptom generation during a stepped hyperinsulinemic hypoglycemic clamp in islet transplant recipients and compare the results with those obtained from type 1 diabetes and nondiabetic control subjects.

	Subjects and Methods

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Subjects

Subjects were recruited from the islet transplantation program at the Hospital of the University of Pennsylvania (HUP). The type 1 diabetic subjects (n = 6) had long-standing C-peptide-negative disease and were on the waiting list for islet transplantation because of frequent severe hypoglycemia complicated by reduced awareness. Three subjects each used multiple daily injections of lispro and glargine insulin or lispro via an insulin pump. Three of these six subjects had a well-functioning kidney allograft supported by 5 mg of daily prednisone, tacrolimus, and either rapamycin or mycophenolate mofetil. The islet transplant recipients (n = 7) were studied a median 6 (range 6–12) months after their last transplant. The procedure for islet transplantation at HUP has been previously reported (17). In short, subjects underwent one to three intraportal islet infusions under daclizumab induction immunotherapy. Five of the seven islet transplant recipients achieved insulin independence, whereas two continued a markedly reduced dose of insulin to maintain near-normal glycemia. Four of these seven subjects had a well-functioning kidney allograft. All received tacrolimus and either rapamycin or mycophenolate mofetil for maintenance immunosuppression, and three subjects with a kidney allograft also received 5 mg of daily prednisone. All medications were withheld on the morning of study.

Healthy nondiabetic control subjects (n = 8) were age, sex, and body mass index (BMI) matched to the islet transplant recipients and type 1 diabetes subjects. This study protocol was approved by the Institutional Review Board of the University of Pennsylvania, and all subjects gave their written informed consent to participate.

Metabolic studies

All subjects were admitted to the HUP General Clinical Research Center the afternoon before study. Subjects fasted overnight after 2000 h for 12 h before testing. Islet transplant recipients who were not insulin independent held any long-acting insulin for longer than 24 h and any rapid-acting insulin for longer than 12 h before testing. Type 1 diabetes subjects similarly held injectable insulin and received iv insulin overnight to maintain the blood glucose concentration 100–150 mg/dl (5.5–8.3 mmol/liter); the insulin infusion was discontinued 30 min before testing. By 0700 h, one catheter was placed in an antecubital vein for infusions and one catheter was placed retrograde in a contralateral hand vein for blood sampling, with the hand placed in a thermoregulated box (50 C) to promote optimal arterialization of venous blood (18). Patency of the iv catheters was maintained with slow infusions of 0.9% saline.

Hypoglycemic clamp

All study participants underwent a modification of the stepped hyperinsulinemic hypoglycemic clamp described by Mitrakou et al. (19). After baseline blood sampling at –30 and 0 min, an insulin infusion was started at 1.0 mU·kg^–1·min^–1 for 270 min followed by 2.0 mU·kg^–1·min^–1 for 90 min. Next, a variable rate infusion of 20% glucose was initiated according to the glycemic clamp technique (20) to achieve 45 min plasma glucose plateaus of 80, 65, 55, and 45 mg/dl at 90-min intervals. Plasma glucose was measured every 5 min at the bedside with a portable glucose analyzer (YSI 1500 Sidekick; Yellow Springs Instruments, Yellow Springs, OH) to adjust the glucose infusion rate and achieve the desired plasma glucose concentration. Additional blood samples were taken every 30 min for biochemical analysis. A questionnaire was administered every 15 min during the study to quantitate autonomic symptoms as the sum of scores ranging from 0 (none) to 10 (severe) for each of the following symptoms: anxiety, palpitations, sweating, tremor, hunger, and tingling (21).

Euglycemic clamp

To calculate the glycemic thresholds for activation of counterregulatory hormone and symptom responses, a hyperinsulinemic euglycemic clamp was performed in randomized order with the hypoglycemic clamp at least 1 wk apart in the control subjects (n = 8) and a subgroup of the islet transplant recipients (n = 4). The euglycemic clamp serves to control for the effects of hyperinsulinemia, time of day, and experimentation on glucagon, cortisol, and symptom responses, respectively. The hyperinsulinemic euglycemic clamp was conducted as described for the hypoglycemic clamp above but with a target plasma glucose of 90 mg/dl (5.0 mmol/liter) for the entire 360-min study (19, 22).

Biochemical analysis

Blood samples were collected on ice into chilled tubes containing EDTA and the protease inhibitors leupeptin and aprotinin (Sigma-Aldrich, St. Louis, MO) for peptide hormones and cortisol and tubes containing EGTA and glutathione (Amersham, Arlington Heights, IL) for catecholamines, centrifuged at 4 C, separated, and frozen at –80 C for subsequent analysis. Plasma immunoreactive insulin, glucagon, GH, and cortisol were measured in duplicate by double-antibody RIA (Linco Research, St. Charles, MO). Plasma epinephrine and norepinephrine were measured by HPLC with electrochemical detection. Samples from paired euglycemic-hypoglycemic experiments of each subject were assayed simultaneously.

Calculations and statistics

The glycemic threshold for a given parameter (i.e. hormonal or symptom response) in each subject was considered to be the plasma glucose concentration (mean of three measurements every 15 min) at which the parameter first consistently exceeded the 95% confidence limit observed for that parameter at the corresponding time point in the euglycemic control experiments, after adjustment of the experimental and control baseline data to zero (19, 23). Because levels of each parameter were not statistically different between the control (n = 8) and islet transplant (n = 4) groups during the euglycemic clamp, data from both groups were combined (n = 12) to determine the 95% confidence limits. If a given parameter in a subject failed to exceed the 95% confidence limit by 360 min, the nadir plasma glucose concentration (mean over the last 15 min of the clamp) was used as the glycemic threshold in that subject for statistical purposes (6). The magnitude of each hormone and symptom response was assessed as the incremental value by subtracting baseline levels (basal) from those obtained during the last 60 min of hypoglycemia (final) (6). All data are expressed as mean ± SE. Intergroup comparisons were made by one-way or repeated-measures ANOVA as appropriate, and when significant differences were found, followed by Fisher’s least significant difference test or where noted, Student’s t test, using Statistica software (Tulsa, OK). Significance was considered at P < 0.05 (two tailed).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject characteristics

All three groups were comparable in age, sex, BMI, and basal glucose levels, whereas the hemoglobin A_1c (HbA_1c) was significantly higher in the type 1 diabetic subjects (P < 0.01 vs. control group), and this group required higher basal insulin levels (P < 0.01 vs. control group) to maintain normoglycemia (Table 1). Whereas basal glucagon levels were comparable across all three groups, basal C-peptide was undetectable in the type 1 diabetes subjects and not different between the islet transplant and control groups (Table 1). The type 1 diabetes subjects and islet transplant recipients shared a comparably long duration of diabetes (30 yr); the islet recipients had received 15,042 ± 1,622 islet equivalents (IEs) per kilogram body weight, required significantly less insulin therapy, and had negligible scores on the survey of Clarke et al. (24) of hypoglycemia severity, compared with the type 1 diabetic subjects (P < 0.001 for both comparisons; Table 1).

During the hypoglycemic clamp, plasma glucose by 90 min in all three groups was near 80 mg/dl (4.4 mmol/liter) and thereafter overlapped during the 65 (135–180 min), 55 (225–270 min), and 45 (315–360 min) mg/dl (3.6, 3.1, and 2.5 mmol/liter, respectively) glycemic plateaus, whereas during the euglycemic clamp, plasma glucose remained between 85 and 90 mg/dl (4.7–5.0 mmol/liter; Fig. 1, left panel). The insulin infusion rates 1.0 mU·kg^–1·min^–1 for 270 min and 2.0 mU·kg^–1·min^–1 for 90 min both resulted in insulin concentrations that were comparable in all three groups during the hypoglycemic clamp and euglycemic control experiments (Fig. 1, right panel).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Plasma glucose (left panel) and insulin (right panel) during the hyperinsulinemic hypoglycemic clamp in nondiabetic control subjects (, n = 8), type 1 diabetic subjects (, n = 6), and islet transplant recipients (•, n = 7). The shaded area represents the means ± SE for data derived from the hyperinsulinemic euglycemic clamp performed on all control subjects and four islet transplant recipients (n = 12).

All counterregulatory hormone and symptom responses occurred at higher glycemic thresholds and were of greater magnitude in the islet transplant, compared with the type 1 diabetes group; however, these differences reached statistical significance only for glucagon, epinephrine, and GH (magnitude only) (Fig. 2 and Table 2). The glycemic thresholds for glucagon and epinephrine secretion in the islet transplant group were 70 ± 7 and 61 ± 4 mg/dl, respectively, not different from normal (74 ± 4 and 66 ± 4 mg/dl, respectively, in the control group), and significantly greater than in the type 1 diabetes group (48 ± 2 and 49 ± 2 mg/dl, P < 0.01 and P < 0.05, respectively). The magnitude of response for glucagon and epinephrine in the islet transplant group, although significantly greater than in the type 1 diabetes group (P < 0.05 for both responses; Table 2 and Fig. 2), remained significantly less than normal (P < 0.01 for glucagon and P < 0.05 for epinephrine; Table 2 and Fig. 2). The magnitude of GH secretion in the islet transplant group was comparable with normal and significantly greater than in the type 1 diabetes group (P < 0.05; Table 2 and Fig. 2).

View larger version (37K): [in this window] [in a new window]   FIG. 2. Incremental counterregulatory hormone and symptom responses during the hyperinsulinemic hypoglycemic clamp in nondiabetic control subjects (, n = 8), type 1 diabetic subjects (, n = 6), and islet transplant recipients (•, n = 7). The shaded area represents the means ± SE for data derived from the hyperinsulinemic euglycemic clamp performed on all control subjects and four islet transplant recipients (n = 12).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

There is no clear explanation for the failure of islet transplantation to fully normalize the magnitude of epinephrine secretion. One possibility is that varying degrees of autonomic neuropathy may exist in subjects with a very long duration of type 1 diabetes. It has been shown that the magnitude of epinephrine secretion during hypoglycemia is less in type 1 diabetic subjects with documented autonomic neuropathy, compared with those without autonomic neuropathy (27, 28). Thus, the near normalization of glycemia and avoidance of hypoglycemia imparted by an islet transplant may reverse a defect attributable to HAAF but may not reverse any structural deterioration in the autonomic innervation of the adrenal medulla, which is required for epinephrine secretion. Consistent with our results, the transplantation of a whole pancreas in subjects with a comparably long duration of type 1 diabetes normalized the glycemic threshold but not magnitude of epinephrine secretion in response to hypoglycemia (29), an effect that persists for as long as 14 yr after transplant (30). Also in agreement, strict avoidance of hypoglycemia in type 1 diabetes subjects can restore the glycemic threshold (5, 6) but not magnitude (5, 9) of the epinephrine response.

In comparison with the transplantation of isolated islets or a whole pancreas graft, strict hypoglycemia avoidance in type 1 diabetes subjects with reduced hypoglycemia awareness requires intense attention to glucose monitoring and insulin administration, is associated with increased [0.5% or more (5, 6, 7, 8, 9)] rather than decreased HbA_1c but obviously avoids the risks associated with transplantation and immunosuppression. Guidance for attempting strict hypoglycemia avoidance should be available from any endocrinologist experienced in the care of patients with type 1 diabetes and so represents an important initial strategy in the management of reduced hypoglycemia awareness. Whereas the improvement in the glycemic threshold for epinephrine secretion that can follow strict hypoglycemia avoidance may be sustained for at least 1 yr, this benefit may be limited to patients with a duration of diabetes that is less than 15 yr (7). Here, a normal glycemic threshold for epinephrine secretion is demonstrated in subjects with approximately 30 yr of type 1 diabetes between 6 and 12 months after islet transplantation. However, under the standard Edmonton protocol for islet transplantation and immunosuppression, less than 80% of recipients may maintain islet graft function beyond 1 yr (10), and in contrast to whole pancreas transplantation, long-term islet graft durability remains uncertain.

We did not detect significant differences across the three study groups in norepinephrine or autonomic symptom responses but are limited in this analysis by the small number of subjects studied and cross-sectional design. Because the nadir plasma glucose concentration was used as the glycemic threshold for subjects lacking any response, the glycemic thresholds for all of the responses in the type 1 diabetes group may actually be lower than is reported here where we kept the plasma glucose 45 mg/dl or greater (2.5 mmol/liter) during the final glycemic plateau. Quantitatively, the norepinephrine and symptom responses were greater in islet transplant than the type 1 diabetes group and overlapped with normal. Importantly, autonomic symptoms were generated at 50 mg/dl or greater (2.8 mmol/liter) in the islet recipients and control subjects, compared with 46 mg/dl or less (2.6 mmol/liter) in the type 1 diabetes subjects, which places the type 1 diabetes subjects at greater risk for cognitive deterioration before the recognition of hypoglycemia warning symptoms (19). Prospective evaluation of a larger number of type 1 diabetes subjects before and after islet transplantation will be needed to confirm these preliminary findings.

GH secretion in response to hypoglycemia was both normalized in the islet transplant recipients and significantly greater than in the type 1 diabetes subjects, whereas cortisol secretion appeared (nonsignificantly) impaired in the islet group similar to the type 1 diabetes group. Data for the adrenocorticotropic hormone response were similar as for cortisol (not shown). Cortisol secretion may have been inhibited by the synthetic glucocorticoid prednisone (5 mg) administered as part of the immunosuppression regimen in three of six type 1 diabetes subjects and four of seven islet transplant recipients studied; however, no differences were apparent in the cortisol response between the type 1 diabetes and transplanted subjects not receiving prednisone (data not shown). Curiously cortisol has not always been shown to improve after strict hypoglycemia avoidance (9). The normal GH response likely results from the avoidance of hypoglycemia after islet transplantation. This restoration of GH secretion suggests recovery of central recognition of hypoglycemia, which is presumably impaired in HAAF (3), and being independent of the autonomic nervous system, would not be affected by some irreversible injury to peripheral nerves. This finding is in agreement with the studies of strict hypoglycemic avoidance in which GH secretion is invariably restored (5, 6, 7, 9). Because GH makes important contributions to glucose counterregulation (31), particularly in settings of impaired glucagon and epinephrine secretion (32), the restored GH secretion in islet transplant recipients may play a crucial role in their defense against hypoglycemia.

In conclusion, glycemic thresholds for activation of counterregulatory hormone and symptom responses appear normal after islet transplantation for long-standing type 1 diabetes, whereas the magnitude of the glucagon and epinephrine responses remain impaired. The recovery of counterregulatory activation at plasma glucose levels that are appropriate for each response supports the concept of HAAF and its reversibility, whereas additional mechanisms affecting glucose counterregulation, such as effects of islet engraftment on glucagon secretion and autonomic neuropathy on epinephrine secretion, may continue to affect the islet transplant recipient. Future studies are required to understand the mechanisms involved in glucagon and epinephrine secretion in islet transplant recipients and determine the contribution of these responses to hepatic glucose production during hypoglycemia as well as their durability over time.

	Acknowledgments

 
We are indebted to the islet transplant recipients and type 1 diabetes subjects for their participation; to the nursing staff of the HUP General Clinical Research Center for their subject care and technical assistance; to Dr. Heather Collins (University of Pennsylvania Diabetes Endocrinology Research Center) for performance of the RIAs; and to Huong-Lan Nguyen for laboratory assistance.

	Footnotes

 
This work was supported by the Juvenile Diabetes Research Foundation and the Public Health Services Research Grants M01-RR00040 (HUP General Clinical Research Center), P30-DK19525 (University of Pennsylvania Diabetes Endocrinology Research Center), U42-RR016600 (HUP Islet Cell Resources Center), and K12-RR017625 (to M.R.R.) from the National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 27, 2006

Abbreviations: BMI, Body mass index; HAAF, hypoglycemia-associated autonomic failure; Hb, hemoglobin; IE, islet equivalent.

Received November 6, 2006.

Accepted December 18, 2006.

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