Hypoglycemic Clamp Studies Research Articles

Pancreas transplantation restores epinephrine response and symptom recognition during hypoglycemia in patients with long-standing type I diabetes and autonomic neuropathy.

Impaired epinephrine secretion and symptom unawareness are characteristic of severe hypoglycemia in individuals with long-standing type I diabetes. Recently, the avoidance of clinical hypoglycemia has been reported to improve epinephrine and symptom responses to hypoglycemia in type I patients. However, the extent to which these defects can be restored in individuals with long-standing type I diabetes and autonomic neuropathy has not been assessed, nor has it been determined whether pancreas transplantation, which not only obviates hypoglycemia but also prevents hyperglycemia, results in the complete recovery of either epinephrine response or symptom awareness during insulin-induced hypoglycemia. We performed stepped hypoglycemic clamp studies in successful pancreas transplantation recipients to assess epinephrine and other counterregulatory hormone responses during hypoglycemia and to determine the degree to which hypoglycemic symptom recognition could be restored. Thirteen pancreas transplant recipients and matched control subjects were studied utilizing stepped hypoglycemic clamp protocol to achieve target glucose levels of 3.9, 3.3, 2.8, and 2.2 mmol/l (70, 60, 50, and 40 mg/dl, respectively). Plasma epinephrine response was significantly greater in healthy control subjects and pancreas transplant patients compared with type I subjects at the glucose plateaus of 3.9, 3.3, and 2.8 mmol/l. However, epinephrine response in pancreas transplant recipients was significantly less than that seen in either healthy control subjects or nondiabetic kidney transplant recipients at each of these glucose plateaus. The magnitude of the epinephrine response in pancreas transplant type I patients did not correlate with either the duration of diabetes, the duration of transplantation, or the measures of autonomic nerve function. Hypoglycemic symptom recognition was significantly greater in pancreas transplant subjects than type I patients and did not differ between pancreas transplant and control groups. No improvement in norepinephrine response was observed after pancreas transplantation, while glucagon responses to hypoglycemia were normalized in pancreas transplant patients. In conclusion, these studies uniquely demonstrate that successful pancreas transplantation improves epinephrine response and normalizes hypoglycemia symptom recognition in patients with long-standing diabetes and established autonomic neuropathy. No correlation was observed between the severity of autonomic neuropathy or the duration of diabetes and the recovery of either the epinephrine or symptom responses to hypoglycemia.

Defective glucagon secretion during sustained hypoglycemia following successful islet allo- and autotransplantation in humans.

Defective glucagon secretion during hypoglycemia is characteristic of long-standing type I diabetes. To determine whether this defect can be corrected by successful intrahepatic islet transplantation, we performed studies of hypoglycemia in four nondiabetic patients with chronic pancreatitis who had undergone total pancreatectomy and successful intrahepatic islet autotransplantation, in two type I diabetic recipients of successful intrahepatic islet allotransplantation, and in matched control subjects. We examined 1) whether intrahepatic islet autotransplantation provides glucagon secretion during prolonged periods of hypoglycemia and 2) whether intrahepatic islet allotransplantation in type I diabetic patients and consequent long-term normoglycemia reestablishes native alpha-cell responses to hypoglycemia. Glucagon secretion was assessed during 3-h hypoglycemic hyperinsulinemic clamp studies. The islet autograft recipients were studied 63 +/- 19 months posttransplant, and all were insulin-independent and normoglycemic (HbA(1c), 5.8 +/- 0.2%). Neither allograft recipient required exogenous insulin and maintained HbA(1c) levels of 5.7 and 6.4% 30 and 34 months posttransplant, respectively. All recipients were normoglycemic (fasting glucose: autograft recipients, 5.6 +/- 0.1 mmol/l; allograft recipient #1, 6.3 mmol/l; allograft recipient #2, 5.8 mmol/l) at the time of study. During hypoglycemia, no increase in glucagon secretion was observed in either the auto- or allotransplant recipients, whereas healthy control subjects and recipients of kidney transplantation had significant increases in glucagon. In contrast, both allo- and autograft recipients had glucagon responses to intravenous arginine. These data uniquely demonstrate that: 1) intrahepatic islet transplant grafts secrete glucagon in response to arginine, but fail to secrete glucagon in response to sustained hypoglycemia; and 2) the restoration of sustained normoglycemia for over 2 years in type I diabetic patients may not reestablish glucagon responses from the native pancreas during hypoglycemia. Transplantation sites other than the liver may be required to achieve normal glucagon secretion from the transplanted islets.

Role of cortisol in the pathogenesis of deficient counterregulation after antecedent hypoglycemia in normal humans.

The aim of this study was to determine the role of increased plasma cortisol levels in the pathogenesis of hypoglycemia-associated autonomic failure. Experiments were carried out on 16 lean, healthy, overnight fasted male subjects. One group (n = 8) underwent two separate, 2-d randomized experiments separated by at least 2 mo. On day 1 insulin was infused at a rate of 1.5 mU/kg per min and 2 h clamped hypoglycemia (53 +/- 2 mg/dl) or euglycemia (93 +/- 3 mg/dl) was obtained during morning and afternoon. The next morning subjects underwent a 2-h hyperinsulinemic (1.5 mU/kg per min) hypoglycemic (53 +/- 2 mg/dl) clamp study. In the other group (n = 8), day 1 consisted of morning and afternoon 2-h clamped hyperinsulinemic euglycemia with cortisol infused to stimulate levels of plasma cortisol occurring during clamped hypoglycemia (53 mg/dl). The next morning (day 2) subjects underwent a 2-h hyperinsulinemic hypoglycemic clamp identical to the first group. Despite equivalent day 2 plasma glucose and insulin levels, steady state epinephrine, norepinephrine, pancreatic polypeptide, glucagon, ACTH and muscle sympathetic nerve activity (MSNA) values were significantly (R < 0.01) blunted after day 1 cortisol infusion compared to antecedent euglycemia. Compared to day 1 cortisol, antecedent hypoglycemia produced similar blunted day 2 responses of epinephrine, norepinephrine, pancreatic polypeptide and MSNA compared to day 1 cortisol. Antecedent hypoglycemia, however, produced a more pronounced blunting of plasma glucagon, ACTH, and hepatic glucose production compared to day 1 cortisol. We conclude that in healthy overnight fasted men (a) antecedent physiologic increases of plasma cortisol can significantly blunt epinephrine, norepinephrine, glucagon, and MSNA responses to subsequent hypoglycemia and (b) these data suggest that increased plasma cortisol is the mechanism responsible for antecedent hypoglycemia causing hypoglycemia associated autonomic failure.

Differential Regulation of Counterregulatory Hormone Secretion and Symptoms During Hypoglycemia in IDDM: Effect of glycemic control

To determine 1) if there was a differential effect of glycemic control on individual counterregulatory hormone responses to hypoglycemia in patients with insulin-dependent diabetes mellitus (IDDM), 2) whether these modifications affect hypoglycemic symptom perception, and 3) if there was a level of glycemic control below which counterregulatory and symptomatic responses to hypoglycemia become consistently altered. We performed hypoglycemic clamp studies on 38 patients with IDDM and 38 healthy control subjects. Glucose was lowered from 5.0 to 2.2 mmol/l over 3 h in decrements of 0.6 mmol/l each 30 min. Epinephrine, cortisol, growth hormone (GH), glucagon, and symptom score were measured at each glucose plateau. In IDDM patients, HbA1 levels were positively correlated with incremental epinephrine (r = 0.58, P < 0.001) and cortisol (r = 0.52, P < 0.01) responses, but inversely correlated with GH responses (r = -0.31, P < 0.05). The increase in symptom score correlated with the epinephrine response in the IDDM patients (r = 0.57, P < 0.001), but not in the healthy control subjects (r = -0.02, NS). In IDDM patients with HbA1 < or = 7.8% (n = 7), the epinephrine, cortisol, and symptomatic responses to hypoglycemia were blunted, but GH secretion was preserved. These data suggest that 1) there is differential regulation of counterregulatory hormone secretion that is dependent on the level of glycemic control, 2) epinephrine is an important determinant of symptom perception in IDDM patients, but not in healthy control subjects, and 3) multiple defects in counterregulatory hormone secretion and symptom perception are consistently observed in patients with HbA1 levels < or = 7.8%.

Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia.

The central nervous system has been implicated in the activation of counterregulatory hormone release during hypoglycemia. However, the precise loci involved are not established. To determine the role of the ventromedial hypoglycemia, we performed hypoglycemic clamp studies in conscious Sprague-Dawley rats with bilateral VMH lesions produced by local ibotenic acid injection 2 wk earlier. Rats with lesions in the lateral hypothalamic area, frontal lobe, sham operated (stereotaxic needle placement into hypothalamus without injection), and naive animals served as control groups. The clamp study had two phases. For the first hour plasma glucose was fixed by a variable glucose infusion at euglycemia (approximately 5.9 mM). Thereafter, for an additional 90 min, glucose was either allowed to fall to (a) mild hypoglycemia (approximately 3.0 mM) or (b) more severe hypoglycemia (approximately 2.5 mM). Glucagon and catecholamine responses of lateral hypothalamic area-, frontal lobe-lesioned, sham operated, and naive animals were virtually identical at each hypoglycemic plateau. In contrast, glucagon, epinephrine, and norepinephrine responses in the VMH-lesioned rats were markedly inhibited; hormones were diminished by 50-60% during mild and by 75-80% during severe hypoglycemia as compared with the other groups. We conclude that the VMH plays a crucial role in triggering the release of glucagon and catecholamines during hypoglycemia.

Gender influences counterregulatory hormone responses to hypoglycemia

It has been generally assumed that counterregulatory hormone responses to hypoglycemia are not influenced by gender. To test this assumption, we analyzed three separate hypoglycemic insulin clamp studies in age-matched, healthy, non-obese females (n = 33) and males (n = 37). In one study (12 females, 17 males), plasma glucose level was rapidly decreased to about 57 mg/dL for 100 minutes with a 0.65-mU/kg/min insulin infusion. Despite an identical decrease in glucose level, the increase in epinephrine (361 ± 64 v 188 ± 38 pg/mL, P < .05), norepinephrine (132 ± 28 v 47 ± 19 pg/mL, P < .01), and growth hormone ([GH] 16.0 ± 3.8 v 4.9 ± 1.9 ng/mL, P < .05) levels, but not glucagon or cortisol levels, were significantly greater in males than in females, respectively. In the second study (10 females, eight males), a 5.0-mU/kg/min insulin infusion was used to decrease glucose levels to 55 mg/dL for 180 minutes. Epinephrine ( P < .05) and GH ( P < .01) responses were greater in males than in females. In a third study (11 females, 12 males), plasma glucose level was gradually decreased to about 50 mg/dL over 240 minutes. Again epinephrine ( P < .01), norepinephrine ( P < .01), GH ( P < .05), and cortisol ( P < .01) responses were nearly twofold greater in males ( P < .01). Multivariate analysis of all 70 subjects identified gender as the most significant factor contributing to the epinephrine ( P < .001) and norepinephrine ( P < .005) responses, and also as a significant contributor to the GH response ( P < .05). Moreover, the glucose levels that triggered an increase in epinephrine, norepinephrine, and cortisol were each about 9 mg/dL higher in males ( P < .05). We conclude that, with respect to males, females have decreased counterregulatory hormonal responses to hypoglycemia. This sexual dimorphism may be in part due to an alteration in the glucose threshold for hormone release.

Physiologic hyperinsulinemia enhances counterregulatory hormone responses to hypoglycemia in IDDM.

We evaluated the effect of physiologic hyperinsulinemia (plasma insulin 329 +/- 62 vs 687 +/- 62 pmol/L) on counterregulatory hormone responses in 8 IDDM subjects studied during a 2-hour hypoglycemic clamp study with an equivalent degree of hypoglycemia (plasma glucose 3.1 +/- 0.1 and 3.0 +/- 0.1 mmol/L, respectively). Plasma epinephrine levels were increased by 71% during the last 60 minutes of hypoglycemia in the high insulin study (840 +/- 180 vs 1440 +/- 310 pmol/L, respectively p = 0.006). In addition, plasma cortisol and norepinephrine were also increased in the high insulin study (by 19% and 24% respectively, p < 0.01, for both). Plasma growth hormone and glucagon concentrations were not altered by high dose insulin infusion. In spite of increased epinephrine secretion, the glucose infusion rate required to maintain glucose was 2-fold greater in the high insulin study, and there was greater suppression of lipolysis in that group. We conclude that hyperinsulinemia may enhance counterregulatory hormone secretion in IDDM.

Loss of Potentiating Effect of Hypoglycemia on the Glucagon Response to Hyperaminoacidemia in IDDM

IDDM subjects lose the ability to release glucagon during hypoglycemia. Because replacement of basal levels of amino acids enhances the glucagon response to hypoglycemia in healthy subjects, we tested whether raising amino acid levels during hypoglycemia could reverse the defective alpha-cell response in IDDM patients. For this purpose, 11 IDDM patients (HbA1 9.4 +/- 0.6%) and 8 healthy, nondiabetic subjects received two hypoglycemic insulin clamp studies (0.8 mU.kg-1 x min-1) in which plasma glucose was clamped at 55 mg/dl (3.08 mM) for 180 min. During one of the studies, an infusion of amino acids was superimposed between 120 and 180 min (0.3 g.kg-1 x h-1). This dose of amino acids had a small effect on plasma glucagon levels during euglycemic hyperinsulinemia that was comparable in normal and IDDM subjects. In healthy control subjects, plasma glucagon rose by 80% during the initial hypoglycemic phase of the study. The addition of amino acids produced a further sharp (200-250 ng/L, P < 0.02) rise in plasma glucagon, such a change did not occur in the absence of amino acids. In contrast, plasma glucagon in IDDM patients failed to increase during hypoglycemia alone and rose by only 40-50 ng/L (P < 0.05 vs. controls) when amino acid infusion was superimposed, even though plasma amino acid levels rose to the same extent in IDDM and control subjects. More importantly, the rise in glucagon produced by amino acids was comparable during hypoglycemic and euglycemic hyperinsulinemia in the IDDM patients, results strikingly different from those observed in nondiabetic control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

SUPPRESSION OF COUNTERREGULATORY HORMONE RESPONSE TO HYPOGLYCEMIA BY INSULIN PER SE

Although assessment of counterregulatory hormone responses to hypoglycemia relies upon insulin to lower the glucose level, it is not known if the exogenous insulin does used itself influences the magnitude of the hormone response. To assess this, 12 normal subjects randomly received 2 hypoglycemic clamp studies in which the only variable was the insulin dose (0.6 or 5.0 mU/kg-min). Despite 10-fold differences in circulating insulin (265 +/- 29 vs 2576 +/- 222 pmol/L respectively), the hypoglycemic stimulus did not vary. Glucose levels fell over one hour, and then were maintained for two hours at the same hypoglycemic plateau (approximately 3.1 mmol/L for each study) by a variable glucose infusion. Although basal counterregulatory hormone levels in low and high dose studies were indistinguishable, during hypoglycemia the response of epinephrine, growth hormone, and glucagon was significantly suppressed when the degree of hyperinsulinemia was increased. We conclude that raising the magnitude of hyperinsulinemia suppresses the magnitude of the counterregulatory hormone response to hypoglycemia in normal subjects. This modulating effect of insulin per se is yet another variable in the interpretation of hypoglycemic counterregulation.

Opiate blockade enhances hypoglycemic counterregulation in normal and insulin-dependent diabetic subjects

To examine the impact of opiate blockade on glucose counterregulation we performed two hypoglycemic insulin clamp studies with and without naloxone in healthy subjects and well-controlled insulin-dependent (IDDM) patients with defective glucose counterregulation. During both studies plasma glucose fell to 55-60 mg/dl and was then maintained at that level using a variable glucose infusion. In normal subjects, naloxone increased glucose production, thereby reducing the exogenous glucose dose needed to maintain the hypoglycemic plateau. Epinephrine and cortisol responses to hypoglycemia were increased during naloxone plus insulin compared with insulin alone; glucagon responses were unaffected. IDDM patients with suppressed hepatic and hormonal responses to insulin-induced hypoglycemia also demonstrated greater stimulation of glucose production as well as epinephrine, growth hormone, and cortisol release during the naloxone study. In the absence of hypoglycemia, naloxone did not significantly affect glucose production or glucoregulatory hormones. We conclude that opiate blockade augments glucoregulatory responses to insulin-induced hypoglycemia, even in IDDM patients with preexisting defects in glucose counterregulation. This effect is at least in part due to enhanced counterregulatory hormone release during hypoglycemia. Endogenous opiates may modulate hormonal responses during hypoglycemia; their blockade could provide a means of ameliorating defective counterregulation in IDDM patients.

Mild Hypoglycemia and Impairment of Brain Stem and Cortical Evoked Potentials in Healthy Subjects

To evaluate the impact of mild hypoglycemia on CNS function in healthy adults, we measured brain stem auditory evoked potentials and P300 potentials (elicited by cognitive processing of auditory stimuli) during hypoglycemic or euglycemic insulin clamps (80 mU.m-2.min-1). In the hypoglycemic clamp study (n = 8), plasma glucose was allowed to fall from 4.6 to 3 mM in hourly approximately 0.5-mM steps and subsequently returned to euglycemic baseline levels. In the euglycemic clamp study (n = 8), plasma glucose was maintained at baseline levels throughout. Neither brain stem nor P300 responses changed during the euglycemic control study; symptoms and counterregulatory hormones were also unaffected. During the hypoglycemia study, epinephrine and growth hormone rose once plasma glucose reached 3.4 +/- 0.1 mM. Brain stem and P300 potentials remained unchanged until the 3-mM glucose step, when neurophysiological changes suddenly developed in conjunction with reported symptoms. At this glucose level, the wave V component of the brain stem potential was selectively altered in 7 of 8 subjects. Furthermore, P300 latency significantly increased, and amplitude diminished. Changes in both brain stem and cortical (P300) responses reversed when euglycemia was restored. We conclude that modest reductions in plasma glucose (to 3 mM) produce marked alterations in both brain stem and cortical responses to auditory stimuli. These changes in neural function appear at the same time as symptoms and follow rather than precede the rise in counterregulatory hormones during hypoglycemia. Our data suggest that the adverse effects of mild hypoglycemia on brain function are not limited to higher centers but also involve the brain stem.

Change in Hexose Distribution Volume and Fractional Utilization of [18F]-2-Deoxy-2-Fluoro-D-Glucose in Brain During Acute Hypoglycemia in Humans

We used positron emission tomography (PET) to study the effects of mild hypoglycemia on cerebral glucose uptake and metabolism. Nine healthy men were studied under basal saline-infusion conditions, and during euglycemic and hypoglycemic clamp studies. Insulin was infused at the same rate (1 mU.kg-1.min-1) in both clamp studies. In euglycemic clamp studies, glucose was infused at a rate sufficient to maintain the basal plasma glucose concentration, whereas in hypoglycemic clamp studies, the glucose infusion rate was reduced to maintain the plasma glucose at 3.1 mM. Each study lasted 3 h and included a 30-min baseline period and a subsequent 150-min period in which insulin or glucose was administered. Blood samples for measurement of insulin, glucose, cortisol, growth hormone, and glucagon were obtained at 20- to 30-min intervals. A bolus injection of 5-10 mCi [18F]-2-deoxy-2-fluoro-D-glucose (2-DFG) was administered 120 min after initiation of the study, and plasma radioactivity and dynamic PET scans were obtained at frequent intervals for the remaining 40-60 min of the study. Cerebral regions of interest were defined, and concentrations of radioactivity were calculated and used in the three-compartment model of 2-DFG distribution described by Sokoloff. Glucose levels were similar during saline-infusion (4.9 +/- 0.1 mM) and euglycemic clamp (4.8 +/- 0.1 mM) studies, whereas the desired degree of mild hypoglycemia was achieved during the hypoglycemic clamp study (3.1 +/- 0.1 mM, P less than 0.05). The insulin level during saline infusion was 41 +/- 7 pM.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma glucose concentrations at the onset of hypoglycemic symptoms in patients with poorly controlled diabetes and in nondiabetics.

We tested the hypothesis that during decrements in plasma glucose concentration, symptoms of hypoglycemia may occur at higher glucose concentrations in patients with poorly controlled insulin-dependent diabetes mellitus than in persons without diabetes. Symptoms of hypoglycemia and counterregulatory neuroendocrine responses were quantified during hypoglycemic and euglycemic clamp studies in eight patients with insulin-dependent diabetes mellitus selected because their hemoglobin A1 levels were above 10 percent. These data were compared with similar observations in 10 nondiabetic subjects studied previously. Glycemic thresholds--the plasma glucose concentrations during each hypoglycemic clamp study at which a given symptom or biochemical measurement first exceeded its 95 percent confidence interval determined in the euglycemic clamp studies--were calculated for each variable. The mean (+/- SE) glycemic threshold for the symptoms of hypoglycemia was 4.3 +/- 0.3 mmol per liter (78 +/- 5 mg per deciliter) in patients with poorly controlled diabetes--significantly higher (P less than 0.001) than the value of 2.9 +/- 0.1 mmol per liter (53 +/- 2 mg per deciliter) in subjects without diabetes. The mean glycemic thresholds for growth hormone, epinephrine, and cortisol secretions were not significantly different in the two groups. Thus, during decreases in the plasma glucose concentration, patients with poorly controlled insulin-dependent diabetes mellitus may experience symptoms of hypoglycemia at higher plasma glucose concentrations than persons without diabetes. The mechanism underlying this observation remains to be defined.

Electroencephalography and visually evoked potentials during moderate hypoglycemia.

The effects of hypoglycemia per se on the electroencephalogram (EEG) and visually evoked potentials (VEPs) were studied in eight normal young adults. The EEG and VEPs were recorded before and during hypoglycemic clamp studies, carried out at plasma insulin and glucose concentrations of about 287 pmol/L and 2.38 mmol/L, respectively. From the mean power EEG spectra obtained during each testing condition, several parameters in each frequency band considered were compared statistically. During the eyes closed recording, the mean frequency of the alpha-band (8-13 Hz) decreased from 10.1 +/- 0.2 (+/- SE) Hz in both the right and left frontal leads during euglycemia to 8.8 +/- 0.2 and 8.8 +/- 0.1 Hz (left and right frontal leads, respectively; P less than 0.05) during hypoglycemia. In the same leads, the peak frequency decreased from 10.6 +/- 0.4 and 10.3 +/- 0.4 Hz to 9.6 +/- 0.4 and 9.5 +/- 0.3 Hz, respectively (P less than 0.05). A similar pattern of variation was found during the eyes open recording. In contrast, mean VEP latencies did not vary significantly; they were 118 +/- 3 ms (smallest image size; square wave signals subtending 30 min of arc) and 116 +/- 3 ms (largest image size; square wave signals subtending 60 min of arc) during euglycemia to 121 +/- 3 and 119 +/- 3 ms, respectively, during hypoglycemia. This study demonstrates that the earliest hypoglycemia-induced EEG alterations occur in the frontal regions and can be quantified in terms of decreased mean and peak frequencies of the alpha-band. VEP latency is less sensitive. If confirmed in diabetic patients, these data may provide a theoretical basis for developing a portable device to detect early hypoglycemia in those patients who lack warning symptoms.