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The Brain and Hypoglycemic Counterregulation: Insights From Hypoglycemic Clamp Studies

Walter P. Borg, MD
Monica A. Borg, MD
William V. Tamborlane, MD

The results of the Diabetes Control and Complication Trial (DCCT) put to rest the longstanding debate over whether intensive insulin therapy can delay the development of late microvascular and neuropathic complications of diabetes.¹ The benefits of intensive insulin therapy aimed at near-normalization of glucose levels in type I diabetes have been established, and, as a result, this therapeutic approach has been recommended as the treatment of choice for most patients with type I diabetes.² Unfortunately, the frequency and severity of hypoglycemia is markedly increased by intensive treatment regimens, in spite of close medical supervision. The DCCT reported a threefold higher incidence of severe hypoglycemia and coma in those patients assigned to intensified therapy as compared to conventionally-treated controls.^1,3

In the past, iatrogenic hypoglycemia in patients with diabetes was attributed to the so-called "conventional risk factors," such as excessive or inappropriately timed insulin doses, skipped meals or snacks, and excessive exercise. However, analysis of the epidemiology of hypoglycemic episodes has demonstrated that these conventional risk factors are responsible for only a minority of cases.³ It is now appreciated that diabetes mellitus is a disease characterized not only by absolute or relative insulin deficiency, but also by the impairment of hormonal responses that counteract insulin-induced hypoglycemia. This discovery has renewed interest in the physiology of counterregulation in general and in the role of central nervous system (CNS) glucose sensors in particular. It has been hoped that in-depth characterization of the effects of diabetes and its treatment on counterregulatory mechanisms will eventually lead to strategies or interventions that would eliminate this major obstacle to the benefits of intensive insulin therapy.

Defective Glucose Counterregulation
in Type I Diabetes
In subjects without diabetes, the earliest and most important response to a fall in blood glucose level is the suppression of endogenous insulin secretion. In individuals with diabetes who are treated with exogenous insulin, there is no feedback suppression of plasma insulin levels, and in the presence of insulin autoantibodies, the hypoglycemic effect of insulin may be prolonged and may impair recovery from hypoglycemia.⁴In those without diabetes, if suppression of insulin secretion is insufficient to stabilize glucose levels, glucagon and epinephrine are the key counterregulatory hormones that counteract the fall in blood glucose.⁵ However, just as diabetes destroys the ability of the pancreatic ß-cell to secrete insulin in response to hyperglycemia, the ability of the a-cell to secrete glucagon in response to hypoglycemia is lost in many patients with established type I diabetes.⁶ By contrast, glucagon responses to other secretagogues, such as amino acids, are unaffected or even increased in patients with diabetes.

In the absence of a glucagon response, epinephrine becomes the most important hormone for promoting glucose recovery. Activation of the autonomic nervous system, including stimulation of epinephrine secretion, serves to alert patients to falling blood glucose levels by inducing the classical warning symptoms and signs: pounding heart, tremors, hunger, and sweating. In individuals without diabetes, epinephrine responses alone are sufficient to promote recovery from hypoglycemia, even when glucagon secretion is blocked by agents such as somatostatin. Unfortunately, diabetes also adversely affects this line of defense against hypoglycemia. Although deficient epinephrine responses are commonly observed in long-standing diabetes, this impairment is not simply a consequence of aging. The epinephrine response to hypoglycemia is similar in healthy elderly subjects and in young adults without diabetes.⁷

It has long been recognized that the degree of diabetes control strongly influences how patients recognize and respond to reductions in plasma glucose levels. Indeed, in his Nobel Lecture delivered in Stockholm on September 15, 1925, Frederick G. Banting noted that:

"The onset of hypoglycemic symptoms depends not only on the extent, but also on the rapidity, of fall in blood sugar. The level at which symptoms occur is slightly higher in the diabetic with marked hyperglycemia than in a patient whose blood sugar is normal. When the blood sugar is suddenly reduced from a high level, premonitory symptoms may occur with a blood sugar between the normal levels of 0.100% and 0.080%, while the more marked symptoms of prostration, perspiration, and incoordination develop between 0.080% and 0.042%. As a patient becomes accustomed to a normal blood sugar, the threshold of these reactions becomes lower. One patient who formerly had premonitory symptoms of hypoglycemia at 0.096% now has no reaction at 0.076%, but symptoms commence between this level and 0.062%."

With the advent of the insulin pump and multiple injection therapy in the late 1970s and early ’80s, it became apparent that even lower plasma glucose levels were required to induce hypoglycemic symptoms in well-controlled, intensively-treated patients with type I diabetes, suggesting that the threshold for release of epinephrine had been lowered to subnormal values.

Hypoglycemic Clamp Technique
One of the major obstacles in studying counterregulatory hormone responses to hypoglycemia was the lack of methods that would allow investigators to produce a controlled and reproducible hypoglycemic stimulus in vivo. Interpretation of the studies in which hypoglycemia was induced by large bolus injections or continuous infusions of insulin was difficult because of the variations in the rate and the magnitude of the glucose decline between subjects and in the same subjects studied under different conditions. To overcome these obstacles, the glucose clamp was modified to provide a standardized hypoglycemic stimulus in which the rate and magnitude of glucose fall were under the control of the investigator. With this technique, subjects are studied in the fasting state, and separate intravenous catheters are inserted for infusion of exogenous glucose and insulin and for obtaining sequential blood samples. Generally, a fixed rate of insulin is administered in a dose sufficient to obtain the desired degree of hypoglycemia, and exogenous glucose is infused at a variable rate to control the rate and degree of fall of plasma glucose. The rate of glucose infusion is adjusted every 5 minutes based on plasma glucose measurements made at the bedside, and the plasma glucose level is allowed to fall in one or more steps over several minutes or several hours.

The hypoglycemic clamp technique was initially used in our laboratory to test the hypothesis, proposed by Banting above, that the rate of glucose decline influences counterregulatory hormone responses to hypoglycemia in normal subjects and conventionally treated subjects with diabetes.⁸ In one group of experiments, plasma glucose was reduced gradually from approximately 90 to 50 mg/dl (4.4 to 2.8 mM) in 10 mg/dl (0.5 mM) steps over 2 hours. In the other experiment, euglycemia was maintained for 2 hours and then plasma glucose was acutely lowered in one step from 90 to 50 mg/dl over 10–15 minutes. Despite the marked differences in the rate at which plasma glucose declined, no significant differences were detected in the magnitude of the elevations of any of the counterregulatory hormones in either healthy volunteers or conventionally-treated subjects with diabetes.⁸ However, the plasma glucose level that triggered the release of epinephrine (determined from the study when plasma glucose was reduced slowly) varied widely between subjects (range: 48–74 mg/dl, or 3.0–4.1 mM). These observations suggested that individual variations in the glucose thresholds for epinephrine release might contribute to differences in the plasma glucose levels associated with hypoglycemic symptoms, as Banting had also indicated.

In two subsequent separate studies, single-step and multiple-step hypoglycemic clamps were used to examine prospectively the effects of strict glycemic control on epinephrine responses to hypoglycemia.^9,10 In these studies, the magnitude of the epinephrine response to identical reductions in plasma glucose was reduced by 50–60% in the patients with type I diabetes after achieving near-normal glycosylated hemoglobin values with continuous subcutaneous insulin infusion, reaching levels that were significantly below those in healthy controls. Epinephrine release was impaired after intensive therapy irrespective of whether glucose was lowered rapidly in one step from 90 to 50 mg/dl9 or gradually in several steps over 4 hours.¹⁰ Indeed, in the latter study, impaired epinephrine release was shown to be due to a downward shift in the glucose level, which triggered the release of epinephrine.¹⁰Delayed release of other counterregulatory hormones (cortisol, growth hormone, and norepinephrine) was also observed with intensive insulin treatment, and defective glucagon responses were not restored. As might be expected from these results, a greater hypoglycemic stimulus was required to elicit signs and symptoms of hypoglycemia when patients were strictly controlled.¹⁰

Because defects in epinephrine release developed after only 2–6 months of intensive therapy in healthy young patients with diabetes who did not have clinical autonomic neuropathy, it was likely that the impairment was due to acclimatization to lower ambient glucose concentrations during intensive management rather than being caused by some other confounding variable. A similar defect in counterregulatory hormone release was subsequently observed in chronically hypoglycemic insulinoma patients and in women with diabetes who were aggressively treated during pregnancy.^11-12

Further clamp studies have demonstrated that a number of other factors besides the degree of metabolic control of diabetes influence the glucose threshold for counterregulatory hormone responses (Table 1).¹³ It is particularly noteworthy that adolescents with and without diabetes have early and exaggerated epinephrine responses compared to those in adults.¹⁴ Never-the-less, in the DCCT,³ being an adolescent was the only other independent risk factor for the development of severe hypoglycemia besides low HbA^1c level. The large doses of insulin that are typically required in adolescent patients and irregularities in diet and exercise undoubtedly contributed to the greater frequency of severe hypoglycemia in this group in the DCCT, even though enhanced counterregulatory responses should have protected adolescents from hypoglycemia.

Awareness of Symptoms
Related to Hypoglycemia
Ten or fifteen years ago, hypoglycemic symptoms were broadly divided into early "adrenergic" warning symptoms and later neuroglycopenic symptoms, if plasma glucose levels continued to fall. By combining the stepped hypoglycemic clamp with more careful scoring of hypoglycemia-related symptoms, new insights have been obtained regarding the hormonal and physiological changes responsible for symptom awareness in subjects with and without diabetes. In subjects without diabetes, Schwartz and colleagues¹⁵ showed that the glycemic threshold for an increase in symptom scores was 53 ± 2 mg/dl (2.9 ± 0.1 mM), significantly lower than the glucose threshold for increases in plasma epinephrine levels (68 ± 2 mg/dl, or 3.8 ± 0.1 mM). In contrast, in adults with poorly-controlled type I diabetes, the glycemic threshold for symptoms of hypoglycemia (77 ± 6 mg/dl, or 4.3 ± 0.3 mM) was higher than that for release of epinephrine,¹⁶ suggesting that adults with poorly-controlled diabetes are able to perceive low blood glucose levels before the adrenergic response is triggered.

Towler and associates¹⁷ quantified hypoglycemia-related symptom scores in young adult volunteers without diabetes under four conditions: 1) clamped euglycemia, 2) clamped hypoglycemia, 3) clamped hypoglycemia with combined a- and ß-adrenergic blockade, i.e., simultaneous administration of phentolamine and propranolol, and 4) clamped hypoglycemia with panautonomic adrenergic and cholinergic blockade with phentolamine, propranolol, and atropine. Subjects’ response to the question of overall awareness of hypoglycemia (i.e., blood glucose low) did not change during euglycemia but did increase during hypoglycemia. Symptom scores that declined significantly during adrenergic blockade included shaky/tremulous, heart pounding, and nervous/anxious, whereas symptoms of being sweaty, hungry, and tingling did not change. This indicated that they were cholinergic. Neuroglycopenic symptoms, i.e., those produced by hypoglycemia but not reduced by panautonomic blockade, included feeling warm or weak, difficulty thinking/confused, and tired/drowsy. The authors concluded that cholinergic mechanisms mediate an important and previously uncharacterized component of the symptoms of hypoglycemia and awareness of hypoglycemia.

Kerr and associates used the clamp technique in a different way to examine individual components of the hypoglycemia-related symptom complex.¹⁸ They compared symptom scores in 10 healthy volunteers during a hypoglycemic clamp with symptom scores in volunteers during a euglycemic clamp combined with exogenous infusion of epinephrine, norepinephrine, cortisol, glucagon, and growth hormone to mimic the plasma hormone profile observed during the hypoglycemic clamp. Although the hormone infusion caused adrenergic symptoms to increase, as they did during the hypoglycemic clamp, hunger, sweating, and "feeling low" did not increase. This suggested that these symptoms are specific to hypoglycemia per se and are not the hormonal action of epinephrine.

Studies examining changes in circulating hormone concentrations during hypoglycemia have emphasized the importance of adrenomedullary rather than central sympathetic stimulation, since increments in plasma epinephrine during hypoglycemia greatly exceed those of norepinephrine. A very different picture emerges, however, when changes in local concentrations of epinephrine and norepinephrine in peripheral tissues are measured. Maggs and colleagues recently accomplished this by combining the stepped hypoglycemic clamp with microdialysis techniques to estimate changes in catecholamine levels in adipose and muscle extracellular fluid during baseline, hyperinsulinemic euglycemia, and hypoglycemic conditions.¹⁹ As expected, plasma catecholamines (unchanged during euglycemia) rose during hypoglycemia, with plasma epinephrine levels increasing about fivefold more than plasma norepinephrine levels. In contrast, at the local tissue level, the hypoglycemia-induced increments in muscle dialysate norepinephrine and epinephrine were nearly identical, suggesting local generation of norepinephrine. Thus, central sympathetic activation, as manifested by local norepinephrine release from the sympathetic nerve endings, may play a more important role in hypoglycemic counterregulation than previously thought.

The Brain and Defective Counterregulation
The hypoglycemic clamp technique has also been used to show that delayed and reduced counterregulatory hormone responses to hypoglycemia induced by intensive treatment may be due to iatrogenic hypoglycemia per se. In healthy subjects and in type I diabetes patients, a brief period of moderate antecedent hypoglycemia reduces hormonal responses and symptoms of hypoglycemia during experimentally induced hypoglycemia on the following day.²⁰ This sequence of events has been called iatrogenic hypoglycemia-associated autonomic failure,²¹ and more recent evidence implicates adaptive alterations in brain glucose transport or metabolism as its pathophysiological basis.

Since the brain is almost exclusively dependent on circulating glucose for its energy needs,²²profound hypoglycemia may cause coma, permanent brain damage,^23-26 or even death.^27-28 It is not surprising, therefore, that the brain would play an important role in hypoglycemia detection and counterregulation in order to protect itself.^29-31 For example, animal studies have shown that maintenance of normal cerebral glucose levels by infusion of glucose into the carotid and vertebral arteries during systemic hypoglycemia markedly reduces the counterregulatory hormone responses observed when CNS, as well as systemic, glucose concentrations were reduced.³² Hence, it has been hypothesized that the lower glucose threshold for counterregulatory hormone release caused by recurrent hypoglycemia in type I diabetes is due to a reciprocal increase in glucose transport across the blood-brain barrier. In non-diabetic rats, recurrent hypoglycemia increases the efficiency of glucose extraction by the brain^33-35 and also is able to induce defects in counterregulatory hormone responses to hypoglycemia that are comparable to those in intensively-treated patients with diabetes.³⁶

Boyle and associates used the hypoglycemic glucose clamp in combination with measurements of cerebral blood flow and brain arteriovenous differences to examine these issues in human subjects. In volunteers without diabetes, they demonstrated that recurrent insulin-induced hypoglycemia for more than 56 hours leads to adaptations that allow maintenance of normal brain glucose uptake and induces defects in counterregulatory responses during mild hypoglycemia. ³⁷Using the same techniques, they subsequently showed that patients with type I diabetes who have nearly normal glycosylated hemoglobin values maintain normal glucose uptake in the brain during hypoglycemia. In turn, such preservation of normal cerebral metabolism may reduce counterregulatory hormone responses and cause unawareness of hypoglycemia.³⁸

It should also be noted that transport and metabolism of glucose may not be the only brain substrate affected by diabetes. Studies that have combined clamps with microdialysis techniques have reported strikingly high concentrations of lactate³⁹ in brain extracellular fluid during hypoglycemia.

Episodes of severe hypoglycemia in children are often followed by reversible unilateral neurological deficits. It has been proposed that hypoglycemia per se may result in the disturbance of the cerebral blood flow sufficient to result in transient focal ischemia. To address this issue, Jarjour and colleagues investigated the effect of mild hypoglycemia on regional cerebral blood flow and cerebrovascular resistance in right-handed children with type I diabetes, using the intravenous xenon-133 clearance method.⁴⁰These results showed that cerebral gray matter blood flow was significantly higher in the right hemisphere compared to the left during hypoglycemia but not at baseline euglycemia. Such asymmetrical cerebral blood flow changes may explain the frequent laterality of neurological deficits after severe hypoglycemia.

Role of the Ventromedial Hypothalamus
The importance of the CNS in hypoglycemic detection and counterregulation has generated interest in determining the precise location of such glucose sensors in the brain. While various nuclei have been implicated, data from animal studies suggest that counterregulatory responses during hypoglycemia are activated, at least in part, via the hypothalamus.^41-43 It has been frequently suggested that the ventromedial hypothalamus (VMH),29 known as a regulator of food intake ("satiety center"),^44-45 contains glucosensitive tissues that could mediate the responses to hypoglycemia.^46-47

This hypothesis was based primarily on the observations that injections of 2-deoxy-glucose into the third ventricle caused hyperglycemia.⁴⁵ Initially, the VMH was considered a sympathetic center, controlling mainly catecholamine secretion in response to hypoglycemia.^41,48 Glucagon responses were thought to be triggered by intraislet rather than CNS mechanisms.^49,50 It has been demonstrated, however, that electric stimulation of the VMH caused an increase in plasma glucagon levels.^29,51,52 Although these studies did not directly address the role of the VMH during hypoglycemia they suggested that the VMH could serve as a key center for the activation of hypoglycemic counterregulation.

Our studies have further established the VMH as a dominant center for sensing of glucopenia. In rats, focal lesioning of the VMH abolishes hormonal response to systemic hypoglycemia.⁵³ Moreover, production of local neuroglycopenia by perfusion of 2-deoxy-glucose directly into the VMH of awake rats triggers the release of counterregulatory hormones in the absence of systemic hypoglycemia.⁵⁴ Conversely, hormonal responses to systemic hypoglycemia could be suppressed by maintaining normal glucose concentrations in the VMH by glucose infusions via stereotaxically placed microdialysis probes.⁵⁵ Selective prevention of hypoglycemia in the VMH, but not in the remainder of the CNS or elsewhere, blocked catecholamine and glucagon responses, thus providing strong evidence for these hypothalamic glucoreceptors. Since preliminary results indicate that counterregulatory hormone responses to systemic hypoglycemia can also be blocked by infusion of lactate into the VMH, the VMH may act as a fuel sensor rather than being only a glucose sensor.⁵⁶

Brain Function and
Recurrent Hypoglycemia
The hypoglycemic clamp has also permitted investigators to examine whether intensive insulin therapy influences the ability of the brain to function in the face of mild to moderate hypoglycemia. This is of paramount clinical importance since adaptive increases in brain glucose transport induced by recurrent mild hypoglycemia could result in a favorable down-shifting of the glucose level at which brain function becomes impaired.

This issue is controversial, since it has been reported that intensively-treated type I diabetes patients are more, rather than less, vulnerable to neuroglycopenia than poorly controlled counterparts, when conventional electroencephalographic (EEG) recordings are used as an endpoint.⁵⁷ Other studies using neuropsychological tests to assess cognitive performance during hypoglycemia in type I diabetes patients have found no change^58,59 or a lowering of the plasma glucose level required to provoke cognitive dysfunction in patients treated intensively.^60-62 It should be emphasized that the various tests of cognitive function used in these studies are likely to assess different brain regions, which may have different glucose requirements.

In a recent study from our laboratory, brain function was assessed electrophysiologically by measuring cortical auditory evoked potentials. The P300 waveform, unlike the EEG, which measures the spontaneous electrical output of the brain, is generated by the active cognitive processing of stimulus information. It is referred to as the P300 potential because the peak amplitude normally occurs about 300 milliseconds after the sensory stimulus. It requires the active participation of the subject and involves higher brain centers, particularly the hippocampus and the parahippocampal gyrus.⁶³

We have compared P300 responses to hypoglycemia in intensively-treated versus conventionally-treated type I diabetes patients and healthy subjects during stepped hypoglycemia. As expected, glucose levels triggering hormonal responses and perception of hypoglycemic symptoms were significantly lower in intensively-treated patients as compared to their poorly-controlled counterparts, and hormonal responses were suppressed as compared to healthy controls. We also found that a greater reduction in plasma glucose was required to alter P300 potentials in the intensively-treated patients as compared to both the conventionally-treated patients with diabetes and the nondiabetic group. Our data are consistent with those of an earlier report by Ziegler and associates⁶⁴ that also utilized the P300 to monitor changes in cognitive function during hypoglycemia in type I diabetes patients. Both studies suggest that intensively-treated patients are less vulnerable to cortical dysfunction during mild to moderate hypoglycemia.

If well-controlled, intensively-treated patients are less vulnerable to cortical dysfunction during mild to moderate hypoglycemia than are poorly-controlled, conventionally-treated patients, why was the frequency of severe hypoglycemia including seizure and coma so much higher in the intensive treatment group in the DCCT?¹ In well-controlled patients with type I diabetes, it is likely that the same molecular mechanisms that lead to enhanced cortical functioning in the face of falling plasma glucose levels also lead to delayed activation of the glucopenic sensing centers in the VMH. When the latter effect predominates during clinical management, plasma glucose falls to levels below those that can be compensated for by changes in cortical metabolism.

Summary

Increased frequency of hypoglycemia has emerged as a serious side effect of intensive insulin therapy aimed at prevention of late diabetic complications. Patients with diabetes are more vulnerable to low blood glucose levels not only because they are unable to synchronize insulin delivery normally with meal ingestion and activity, but also because they have defective counterregulatory responses to protect them against hypoglycemia.

Recently the role of CNS in activation and coordination of the counterregulatory responses to hypoglycemia has received substantial attention. Many specific techniques have been developed to investigate the intricate relationships between the brain and glucose homeostasis. Among them, the euglycemic-hypoglycemic clamp has been proven to be an effective tool in clarifying the role of the CNS in glucose counterregulation, as well as in investigating the impact of hypoglycemia on brain function. It appears that intensive treatment of type I diabetes leads to adaptive changes that enhance substrate availability for cognitive function, but unfortunately this leads to delayed activation of brain centers that initiate counterregulatory hormone responses. While our understanding of these issues has advanced considerably, strategies to prevent the development of hypoglycemia in insulin-treated patients remain elusive.

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Walter P. Borg, MD, and Monica A. Borg, MD, are post-doctoral fellows in the Department of Internal Medicine, and William V. Tamborlane, MD, is a professor of pediatrics and director of the Children's Clinical Research Center at the Yale University School of Medicine, in New Haven, Conn.