Counterregulatory responses to postprandial hypoglycemia after Roux-en-Y gastric bypass

BackgroundPostbariatric hypoglycemia (PBH) is a potentially serious complication after Roux-en-Y gastric bypass (RYGB), and impaired counterregulatory hormone responses have been suggested to contribute to the condition.ObjectivesWe evaluated counterregulatory responses during postprandial hypoglycemia in individuals with PBH who underwent RYGB.SettingUniversity hospital.MethodsEleven women with documented PBH who had RYGB underwent a baseline liquid mixed meal test (MMT) followed by 5 MMTs preceded by treatment with (1) acarbose 50 mg, (2) sitagliptin 100 mg, (3) verapamil 120 mg, (4) liraglutide 1.2 mg, and (5) pasireotide 300 μg. Blood was collected at fixed time intervals. Plasma and serum were analyzed for glucose, insulin, glucagon, epinephrine, norepinephrine, pancreatic polypeptide (PP), and cortisol.ResultsDuring the baseline MMT, participants had nadir blood glucose concentrations of 3.3 ± .2 mmol/L. At the time of nadir glucose, there was a small but significant increase in plasma glucagon. Plasma epinephrine concentrations were not increased at nadir glucose but were significantly elevated by the end of the MMT. There were no changes in norepinephrine, PP, and cortisol concentrations in response to hypoglycemia. After treatment with sitagliptin, 8 individuals had glucose nadirs <3.2 mmol/L (versus 4 individuals at baseline), and significant increases in glucagon, PP, and cortisol responses were observed.ConclusionsIn response to postprandial hypoglycemia, individuals with PBH who underwent RYGB only had minor increases in counterregulatory hormones, while larger hormone responses occurred when glucose levels were lowered during treatment with sitagliptin. The glycemic threshold for counterregulatory activation could be altered in individuals with PBH, possibly explained by recurrent hypoglycemia.     

Effect of antecedent hypoglycemia on counterregulatory responses to subsequent euglycemic exercise in type 1 diabetes

Exercise-related hypoglycemia is common in intensively treated patients with type 1 diabetes. The underlying mechanisms are not clearly defined. In nondiabetic subjects, hypoglycemia blunts counterregulatory responses to subsequent exercise. It is unknown whether this also occurs in type 1 diabetes. Therefore, the goal of this study was to test the hypothesis that prior hypoglycemia could result in acute counterregulatory failure during subsequent exercise in type 1 diabetes. A total of 16 type 1 diabetic patients (8 men and 8 women, HbA(1c) 7.8 +/- 0.3%) were investigated during 90 min of euglycemic cycling exercise, following either two 2-h periods of previous-day hypoglycemia (2.9 mmol/l) or previous-day euglycemia. Patients' counterregulatory responses (circulating levels of counterregulatory hormones, intermediary metabolites, substrate flux via indirect calorimetry, tracer-determined glucose kinetics, and cardiovascular measurements) were comprehensively assessed during exercise. Identical euglycemia and basal insulin levels were successfully maintained during all exercise studies, regardless of blood glucose levels during the previous day. After resting euglycemia, patients displayed normal counterregulatory responses to exercise. Conversely, when identical exercise was repeated after hypoglycemia, the glucagon response to exercise was abolished, and the epinephrine, norepinephrine, cortisol, endogenous glucose production, and lipolytic responses were reduced by 40-80%. This resulted in a threefold increase in the amount of exogenous glucose needed to maintain euglycemia during exercise. Our results demonstrate that antecedent hypoglycemia, in type 1 diabetes, can produce acute counterregulatory failure during a subsequent episode of prolonged moderate-intensity exercise. The metabolic consequence of the blunted neuroendocrine and autonomic nervous system counterregulatory responses was an acute failure of endogenous glucose production to match the increased glucose requirements during exercise. These data indicate that counterregulatory failure may be a significant in vivo mechanism responsible for exercise-associated hypoglycemia in type 1 diabetes.     

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.     

Lactate-Induced Release of GABA in the Ventromedial Hypothalamus Contributes to Counterregulatory Failure in Recurrent Hypoglycemia and Diabetes

Suppression of GABAergic neurotransmission in the ventromedial hypothalamus (VMH) is crucial for full activation of counterregulatory responses to hypoglycemia, and increased γ-aminobutyric acid (GABA) output contributes to counterregulatory failure in recurrently hypoglycemic (RH) and diabetic rats. The goal of this study was to establish whether lactate contributes to raising VMH GABA levels in these two conditions. We used microdialysis to deliver artificial extracellular fluid or l-lactate into the VMH and sample for GABA. We then microinjected a GABA_A receptor antagonist, an inhibitor of lactate transport (4CIN), or an inhibitor of lactate dehydrogenase, oxamate (OX), into the VMH prior to inducing hypoglycemia. To assess whether lactate contributes to raising GABA in RH and diabetes, we injected 4CIN or OX into the VMH of RH and diabetic rats before inducing hypoglycemia. l-lactate raised VMH GABA levels and suppressed counterregulatory responses to hypoglycemia. While blocking GABA_A receptors did not prevent the lactate-induced rise in GABA, inhibition of lactate transport or utilization did, despite the presence of lactate. All three treatments restored the counterregulatory responses, suggesting that lactate suppresses these responses by enhancing GABA release. Both RH and diabetic rats had higher baseline GABA levels and were unable to reduce GABA levels sufficiently to fully activate counterregulatory responses during hypoglycemia. 4CIN or OX lowered VMH GABA levels in both RH and diabetic rats and restored the counterregulatory responses. Lactate likely contributes to counterregulatory failure in RH and diabetes by increasing VMH GABA levels.Hypoglycemia has long been considered the major limiting factor to attaining proper glycemic control in diabetic patients intensively treated with insulin. Both prior exposure to hypoglycemia and diabetes per se can diminish the glucagon and epinephrine responses that normally correct a fall in blood glucose. While the mechanism(s) responsible for these defects is not well understood, our previous work has shown that the inhibitory neurotransmitter γ-aminobutyric acid (GABA) within the ventromedial hypothalamus (VMH) plays an important role in suppressing glucose counterregulatory responses under these conditions (1). During euglycemia and hyperglycemia, GABA tonically suppresses the release of glucagon and epinephrine, whereas during hypoglycemia, a decrease in VMH GABA output is crucial to allow full activation of the counterregulatory defense responses (2). More importantly, both antecedent hypoglycemia and diabetes lead to increases in GABAergic tone within the VMH that likely contribute to counterregulatory failure (3,4). While these studies demonstrate increased GABAergic tone in the VMH as one potential contributor to counterregulatory failure under these two conditions, the mechanism(s) underlying pathogenesis of the rise in GABA has not been identified.Several theories have been proposed to explain the phenomenon of counterregulatory failure. One of these postulates is that the brain may adapt to using alternative fuel substrates such as ketones or lactate to sustain its metabolic needs during periods of glucose deprivation and that this, in turn, may prevent it from detecting the onset of hypoglycemia (5,6). Astrocytes play a key role in brain energy metabolism and in maintaining the blood-brain barrier, vascular reactivity, regulation of extracellular glutamate levels, and protection from reactive oxygen species—among other functions (7–9). Over a decade ago, Magistretti and colleagues (10,11) proposed the astrocyte-neuron lactate shuttle hypothesis, which posits that astrocytes provide adjacent neurons with lactate during periods of increased activation to help sustain increased metabolic demands. Lactate can be produced by astrocytes through aerobic glycogenolysis when glucose levels are low or it can be generated from the direct catabolism of glucose when glucose levels are high. As such, it may serve as a key substrate under both glucoprivic and insulin-deficient hyperglycemic conditions, making it an ideal candidate for stimulating or sustaining GABAergic neuronal activation in the VMH after recurrent exposure to hypoglycemia or in diabetes. In support of this, ¹³C-magnetic resonance spectroscopy studies conducted in type 1 diabetic patients showed that these individuals had a greater propensity to take up and use monocarboxylic acids compared with their nondiabetic counterparts (12). In a similar manner, studies in intact rats showed that acute hypoglycemia decreases expression of the neuronal lactate transporter, monocarboxylic acid transporter (MCT)2, in the VMH, but recurrent exposure to hypoglycemia prevents this drop (13). These studies indicate that both diabetes and recurrent hypoglycemia can alter brain metabolism in a manner that favors the use of alternative fuel substrates, such as lactate, when glucose supplies become scarce. Hence, the use of alternative fuel substrates may preclude the brain from detecting a fall in plasma glucose levels, and therefore, it may continue to activate mechanisms that suppress glucose counterregulatory responses to hypoglycemia. That local delivery of lactate into the VMH was able to suppress the counterregulatory responses to hypoglycemia is consistent with this hypothesis, although the underlying mechanism was never established (14).In the current study, we examined whether the use of alternate fuel substrates such as lactate by VMH GABAergic neurons can increase GABA output and, subsequently, lead to counterregulatory failure. We showed that lactate dramatically raises extracellular GABA levels in the VMH and suppresses the counterregulatory response in normal, nondiabetic animals. More importantly, our studies show that lactate also serves a pivotal role in stimulating GABA release in the VMH and in the subsequent development of counterregulatory failure in both recurrently hypoglycemic and poorly controlled diabetic animals.     

Comparison of glucose counterregulation during short-term and prolonged hypoglycemia in normal humans

To compare glucose counterregulatory mechanisms during short-term hypoglycemia and prolonged hypoglycemia, insulin was infused either intravenously (160 mU X M-2 X min) for 10 min or subcutaneously (15 mU X M-2 X min) for 12 h in normal volunteers. With each type of insulin infusion, hypoglycemia (approximately 50 mg/dl) was either allowed to develop or was prevented (control experiments) by the glucose-clamp technique. During prolonged hypoglycemia, both increased glucose production (1.55 +/- 0.05 versus 0.33 +/- 0.14 mg X kg-1 X min in control experiments at 12 h, P less than 0.01) and suppressed glucose utilization (1.55 +/- 0.06 versus 3.17 +/- 0.15 mg X kg-1 X min in control studies at 12 h, P less than 0.01) were involved in counterregulation. During short-term hypoglycemia, only increased glucose production (3.23 +/- 0.33 versus 0.06 +/- 0.03 mg X kg-1 X min in control experiments at 60 min) was involved, since glucose clearance actually increased (3.99 +/- 0.20 versus 2.88 +/- 0.02 ml X kg-1 X min in control experiments at 60 min, P less than 0.01). Estimated portal venous insulin concentrations decreased 40% (basal 24 +/- 3 versus 14 +/- 1 mU/ml at 60 min, P less than 0.01) in the short-term hypoglycemia experiments but remained at basal levels (basal 25 +/- 1 versus approximately 26 microU/min between 1 and 12 h) during prolonged hypoglycemia. Despite the fact that hypoglycemia was more gradually induced in the prolonged hypoglycemia model, peak counterregulatory hormone responses were at least as great as those during short-term hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction in SGLT1 mRNA Expression in the Ventromedial Hypothalamus Improves the Counterregulatory Responses to Hypoglycemia in Recurrently Hypoglycemic and Diabetic Rats

The objective of this study was to determine whether the sodium-glucose transporter SGLT1 in the ventromedial hypothalamus (VMH) plays a role in glucose sensing and in regulating the counterregulatory response to hypoglycemia, and if so, whether knockdown of in the VMH can improve counterregulatory responses to hypoglycemia in diabetic rats or rats exposed to recurrent bouts of hypoglycemia (RH). Normal Sprague-Dawley rats as well as RH or streptozotocin (STZ)-diabetic rats received bilateral VMH microinjections of an adenoassociated viral vector containing either the SGLT1 short hairpin RNA (shRNA) or a scrambled RNA sequence. Subsequently, these rats underwent a hypoglycemic clamp to assess hormone responses. In a subgroup of rats, glucose kinetics was determined using tritiated glucose. The shRNA reduced VMH SGLT1 expression by 53% in nondiabetic rats, and this augmented glucagon and epinephrine responses and hepatic glucose production during hypoglycemia. Similarly, SGLT1 knockdown improved the glucagon and epinephrine responses in RH rats and restored the impaired epinephrine response to hypoglycemia in STZ-diabetic animals. These findings suggest that SGLT1 in the VMH plays a significant role in the detection and activation of counterregulatory responses to hypoglycemia. Inhibition of SGLT1 may offer a potential therapeutic target to diminish the risk of hypoglycemia in diabetes.     

Abnormal glucose counterregulation in insulin-dependent diabetes mellitus. Interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion

To evaluate the roles of counterregulatory hormones and insulin antibodies in the impairment of plasma glucose recovery from hypoglycemia in diabetes mellitus, and to assess the relationship between the glucagon response and duration of the disease, 21 insulin-dependent diabetic patients and 10 nondiabetic subjects were studied. The diabetics consisted of 5 patients with recent onset of diabetes (less than 1 mo); 11 with 2.6 +/- 0.3 (mean +/- SEM) yr duration of diabetes, 5 of whom had insulin antibodies; and 5 patients with long-term diabetes (21 +/- 3 yr), insulin antibodies, and autonomic neuropathy. During insulin-induced hypoglycemia (28 mU/m2 X min for 60 min) in patients with recent-onset diabetes, plasma free insulin, glucose, and counterregulatory hormone concentrations did not differ from those of nondiabetic subjects. In patients with insulin antibodies, the disappearance of insulin after insulin infusion was delayed, and both restitution of normoglycemia and plasma glucagon response were blunted compared with patients without antibodies. When glucagon was infused (80-130 ng/m2 X min) during hypoglycemia in diabetics with impaired glucagon responses in order to simulate normal glucagon responses, plasma glucose recovery was normalized in patients without antibodies but not in those with antibodies. In patients with long-standing diabetes, restitution of normoglycemia was further impaired and this was associated with an absent plasma glucagon response and a diminished plasma epinephrine response. Plasma glucagon responses to hypoglycemia were inversely correlated to the duration of diabetes (r = -0.943; P less than 0.0005). It is concluded that impaired A-cell secretion is the predominant mechanism for the delayed glucose recovery after hypoglycemia in diabetic patients without insulin antibodies and normal epinephrine responses. Slowed disappearance of insulin due to the presence of insulin antibodies further delays the restoration of normoglycemia. Patients with long-standing diabetes and autonomic neuropathy exhibit decreased epinephrine secretion, which leads to an additional retardation of glucose recovery. Since plasma glucagon and epinephrine responses to hypoglycemia were normal at the onset of diabetes but diminished in long-term diabetes, it appears that the impaired glucagon and epinephrine responses to hypoglycemia are acquired defects that develop subsequent to B-cell failure.     

Insulin signaling in the central nervous system is critical for the normal sympathoadrenal response to hypoglycemia

Hypoglycemia, hypoglycemia unawareness, and impaired counterregulation are major challenges to the intensive management of type 1 diabetes. While the counterregulatory response to hypoglycemia is predominantly determined by the degree and duration of hypoglycemia, there is now evidence that insulin per se may influence the counterregulatory response to hypoglycemia. To define the role of insulin action in the central nervous system in regulating the counterregulatory response to hypoglycemia, mice with a brain/neuron-specific insulin receptor knockout (NIRKO) and littermate controls were subjected to 90-min hyperinsulinemic (20 mU x kg(-1) x min(-1)) -hypoglycemic (approximately 1.5 mmol/l) clamps. In response to hypoglycemia, epinephrine levels rose 5.7-fold in controls but only 3.5-fold in NIRKO mice. Similarly, in response to hypoglycemia, norepinephrine levels rose threefold in controls, but this response was almost completely absent in NIRKO mice. In contrast, glucagon and corticosterone responses to hypoglycemia were similar in both groups. Thus, insulin action in the brain is critical for full activation of the sympathoadrenal response to hypoglycemia, and altered neural insulin signaling could contribute to defective glucose counterregulation in diabetes.     

Importance of insulin in subjective, cognitive, and hormonal responses to hypoglycemia in patients with IDDM

Not all episodes of hypoglycemia are recognized as such by diabetic patients, suggesting that it is possible for them to adapt to a low blood glucose level, although the mechanism involved is not known. The aim of this study was to examine whether insulin has an effect, independent of blood glucose, on the subjective, cognitive, and hormonal responses to hypoglycemia. Nine patients with insulin-dependent diabetes mellitus (IDDM) participated in three hyperinsulinemic glucose-clamp studies. After 60 min at 4.5 mM, blood glucose was randomized to be 1) maintained at 4.5 mM for 240 min, 2) lowered to 2.8 mM for 180 min followed by 60 min at 2 mM with an insulin infusion rate of 40 mU.m-2.m-1, and 3) fitted to the same protocol as 2 but with an infusion rate of 120 mU.m-2.min-1. Symptoms and awareness of hypoglycemia (100-mm visual analogue scales), cognitive function, and counterregulatory hormone levels were assessed every 30 min. There were no subjective or cognitive changes during the euglycemic study. Awareness and hypoglycemic symptoms (hunger, facial flushing, trembling, and sweating) were attenuated by the higher insulin infusion rate (P less than 0.05 and P less than 0.01, respectively). Cognition was significantly impaired after 60 min at 2.8 mM (P less than 0.001) and deteriorated further when the blood glucose level was lowered to 2 mM (P less than 0.01). Levels of cortisol (P less than 0.01) and growth hormone (P less than 0.05) but not epinephrine were suppressed by the higher insulin infusion rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in regional brain (18)F-fluorodeoxyglucose uptake at hypoglycemia in type 1 diabetic men associated with hypoglycemia unawareness and counter-regulatory failure

We examined the effects of acute moderate hypoglycemia and the condition of hypoglycemia unawareness on regional brain uptake of the labeled glucose analog [(18)F]fluorodeoxyglucose (FDG) using positron emission tomography (PET). FDG-PET was performed in diabetic patients with (n = 6) and without (n = 7) hypoglycemia awareness. Each patient was studied at plasma glucose levels of 5 and 2.6 mmol/l, applied by glucose clamp techniques, in random order. Hypoglycemia-unaware patients were asymptomatic during hypoglycemia, with marked attenuation of their epinephrine responses (mean [+/- SD] peak of 0.77 +/- 0.39 vs. 7.52 +/- 2.9 nmol/l; P < 0.0003) and a reduced global brain FDG uptake ([mean +/- SE] 2.592 +/- 0.188 vs. 2.018 +/- 0.174 at euglycemia; P = 0.027). Using statistical parametric mapping (SPM) to analyze images of FDG uptake, we identified a subthalamic brain region that exhibited significantly different behavior between the aware and unaware groups. In the aware group, there was little change in the normalized FDG uptake in this region in response to hypoglycemia ([mean +/- SE] 0.654 +/- 0.016 to 0.636 +/- 0.013; NS); however, in the unaware group, the uptake in this region fell from 0.715 +/- 0.015 to 0.623 +/- 0.012 (P = 0.001). Our data were consistent with the human hypoglycemia sensor being anatomically located in this brain region, and demonstrated for the first time a change in its metabolic function associated with the failure to trigger a counter-regulatory response.     

Hypothalamic ATP-sensitive K + channels play a key role in sensing hypoglycemia and triggering counterregulatory epinephrine and glucagon responses

It has been postulated that specialized glucose-sensing neurons in the ventromedial hypothalamus (VMH) are able to detect falling blood glucose and trigger the release of counterregulatory hormones during hypoglycemia. The molecular mechanisms used by glucose-sensing neurons are uncertain but may involve cell surface ATP-sensitive K(+) channels (K(ATP) channels) analogous to those of the pancreatic beta-cell. We examined whether the delivery of sulfonylureas directly into the brain to close K(ATP) channels would modulate counterregulatory hormone responses to either brain glucopenia (using intracerebroventricular 5-thioglucose) or systemic hypoglycemia in awake chronically catheterized rats. The closure of brain K(ATP) channels by global intracerebroventricular perfusion of sulfonylurea (120 ng/min glibenclamide or 2.7 microg/min tolbutamide) suppressed counterregulatory (epinephrine and glucagon) responses to brain glucopenia and/or systemic hypoglycemia (2.8 mmol/l glucose clamp). Local VMH microinjection of a small dose of glibenclamide (0.1% of the intracerebroventricular dose) also suppressed hormonal responses to systemic hypoglycemia. We conclude that hypothalamic K(ATP) channel activity plays an important role in modulating the hormonal counterregulatory responses triggered by decreases in blood glucose. Our data suggest that closing of K(ATP) channels in the VMH (much like the beta-cell) impairs defense mechanisms against glucose deprivation and therefore could contribute to defects in glucose counterregulation.     

Effects of Antecedent GABAA Activation With Alprazolam on Counterregulatory Responses to Hypoglycemia in Healthy Humans

OBJECTIVE

To date, there are no data investigating the effects of GABA_A activation on counterregulatory responses during repeated hypoglycemia in humans. The aim of this study was to determine the effects of prior GABA_A activation using the benzodiazepine alprazolam on the neuroendocrine and autonomic nervous system (ANS) and metabolic counterregulatory responses during next-day hypoglycemia in healthy humans.

RESEARCH DESIGN AND METHODS

Twenty-eight healthy individuals (14 male and 14 female, age 27 ± 6 years, BMI 24 ± 3 kg/m², and A1C 5.2 ± 0.1%) participated in four randomized, double-blind, 2-day studies. Day 1 consisted of either morning and afternoon 2-h hyperinsulinemic euglycemia or 2-h hyperinsulinemic hypoglycemia (2.9 mmol/l) with either 1 mg alprazolam or placebo administered 30 min before the start of each clamp. Day 2 consisted of a single-step hyperinsulinemic-hypoglycemic clamp of 2.9 mmol/l.

RESULTS

Despite similar hypoglycemia (2.9 ± 1 mmol/l) and insulinemia (672 ± 108 pmol/l) during day 2 studies, GABA_A activation with alprazolam during day 1 euglycemia resulted in significant blunting (P < 0.05) of ANS (epinephrine, norepinephrine, muscle sympathetic nerve activity, and pancreatic polypeptide), neuroendocrine (glucagon and growth hormone), and metabolic (glucose kinetics, lipolysis, and glycogenolysis) counterregulatory responses. GABA_A activation with alprazolam during prior hypoglycemia caused further significant (P < 0.05) decrements in subsequent glucagon, growth hormone, pancreatic polypeptide, and muscle sympathetic nerve activity counterregulatory responses.

CONCLUSIONS

Alprazolam activation of GABA_A pathways during day 1 hypoglycemia can play an important role in regulating a spectrum of key physiologic responses during subsequent (day 2) hypoglycemia in healthy man.Hypoglycemia continues to be the major limiting factor to good glycemic control in patients with diabetes. During the last two decades, there have been many studies demonstrating that antecedent hypoglycemia can blunt counterregulatory responses to subsequent hypoglycemia in healthy and type 1 and type 2 diabetic individuals (1). Despite the clinical importance and many elegant studies addressing this topic, there remain gaps in our knowledge regarding the mechanisms regulating neuroendocrine and autonomic nervous system (ANS) responses during episodes of repeated hypoglycemia in man.The three major acute neuroendocrine/ANS counterregulatory defenses against a falling plasma glucose include release of glucagon and epinephrine combined with inhibition of endogenous insulin release. All of these mechanisms either fail (i.e., insulin modulation and glucagon release within ∼5 years of type 1 diabetes duration) or become substantially reduced with disease duration (type 2 diabetes). Furthermore, repeated hypoglycemia has been demonstrated to reduce epinephrine and glucagon responses, which are important defenses against subsequent falling blood glucose levels in both type 1 (epinephrine) and type 2 (epinephrine and glucagon) diabetes (2).For many years, the problem of severe or frequent hypoglycemia was thought to be confined almost exclusively to type 1 diabetes. Recent multicenter trials aimed at improving glycemic control both within hospitals and in the community have identified excess adverse events and death plausibly related to hypoglycemia in type 2 diabetes (3,4). The glucagon response to hypoglycemia is initially relatively preserved in type 2 diabetes (although there is decrease with disease duration) (5). However, as the prevalence of hypoglycemia is increasing in type 2 diabetes, it continues to be of importance to understand the mechanisms regulating release of both glucagon and epinephrine during repeated episodes of hypoglycemia.γ-Aminobytyric acid (GABA) is a major inhibitory neurotransmitter. Previous studies have demonstrated increases in GABAergic tone within the ventromedial hypothalamus in rats with repeated hypoglycemia, which is associated with blunted glucagon and epinephrine responses (6). Chan et al. (7) have also demonstrated that blockade of GABA_A receptors within the ventromedial hypothalamus in rats results in increased glucagon and epinephrine responses during hypoglycemia. Studies investigating the effects of GABA_A modulation on counterregulatory responses during hypoglycemia in humans are scarce. In fact, previous studies have used activation of GABA_A receptors rather than changes in GABA concentrations to investigate the role of GABAergic pathways in ANS and neuroendocrine counterregulatory responses during hypoglycemia in humans and primates. van Vugt et al. (8) demonstrated that alprazolam (a potent pharmacologic activator of the benzodiazepine-GABA_A receptor) can inhibit anterior pituitary neuroendocrine responses during acute hypoglycemia in rhesus monkeys. Giordano et al. (9) reported that alprazolam also reduced neuroendocrine and epinephrine responses to acute intravenous insulin bolus–induced hypoglycemia in healthy humans. Breier et al. (10), using a model of 2-deoxyglycose–induced glucoprivic stress in humans, also demonstrated that alprazolam blunted ACTH and epinephrine responses during neuroglycopenia. Lastly, Smith et al. (11), using modafinil to acutely lower GABA levels during clamped hypoglycemia in healthy humans, reported increased heart rate and improved cognitive function with the drug. Thus, available data would indicate that GABA_A activation can acutely reduce, whereas GABA_A blockade can increase, neuroendocrine and sympathoadrenal responses to hypoglycemia. However, it is unknown whether GABA_A activation can play a mechanistic role in causing neuroendocrine and ANS failure during repeated hypoglycemia in healthy humans. Therefore, in the present study, we have tested the hypothesis that antecedent pharmacologic activation of benzodiazepine-GABA_A receptors with alprazolam can result in counterregulatory failure during next-day hypoglycemia in healthy humans.     

Effects of antecedent hypoglycemia on subsequent counterregulatory responses to exercise

Antecedent hypoglycemia can blunt counterregulatory responses to subsequent hypoglycemia. It is uncertain, however, if prior hypoglycemia can blunt counterregulatory responses to other physiologic stresses. The aim of this study, therefore, was to determine whether antecedent hypoglycemia attenuates subsequent neuroendocrine and metabolic responses to exercise. Sixteen lean, healthy adults (eight men and eight women, ages 28+/-2 years, BMI 22+/-1 kg/m2, VO2max 43+/-3 ml x kg(-1) x min(-1)) were studied during 2-day protocols on two randomized occasions separated by 2 months. On day 1, subjects underwent morning and afternoon 2-h hyperinsulinemic (528+/-30 pmol/l) glucose clamp studies of 5.3+/-0.1 mmol/l (euglycemic control) or 2.9+/-0.1 mmol/l (hypoglycemic study). On day 2, subjects underwent 90 min of exercise on a static cycle ergometer at 80% of their anaerobic threshold (approximately 50% VO2max). Glycemia was equated during day 2 exercise studies via an exogenous glucose infusion. Day 1 hypoglycemia had significant effects on neuroendocrine and metabolic responses during day 2 exercise. The usual exercise-induced reduction in insulin, together with elevations of plasma epinephrine, norepinephrine, glucagon, growth hormone, pancreatic polypeptide, and cortisol levels, was significantly blunted after day 1 hypoglycemia (P<0.01). Commensurate with reduced neuroendocrine responses, key metabolic counterregulatory mechanisms of endogenous glucose production (EGP), lipolytic responses, and ketogenesis were also significantly attenuated (P<0.01) after day 1 hypoglycemia. Significantly greater rates of glucose infusion were required to maintain euglycemia during exercise after day 1 hypoglycemia compared with day 1 euglycemia (8.8+/-2.2 vs. 0.6+/-0.6 micromol x kg(-1) x min(-1); P<0.01). During the first 30 min of exercise, day 1 hypoglycemia had little effect on EGP, but during the latter 60 min of exercise, day 1 hypoglycemia was associated with a progressively smaller increase in EGP compared with day 1 euglycemia. Thus, by 90 min, the entire exercise-induced increment in EGP (8.8+/-1.1 micromol x kg(-1) x min(-1)) was abolished by day 1 hypoglycemia. We conclude that 1) antecedent hypoglycemia results in significant blunting of essential neuroendocrine (glucagon, insulin, catecholamines) and metabolic (endogenous glucose production, lipolysis, ketogenesis) responses to exercise; 2) antecedent hypoglycemia may play a role in the pathogenesis of exercise-related hypoglycemia in type 1 diabetic patients; and 3) antecedent hypoglycemia can blunt counterregulatory responses to other physiologic stresses in addition to hypoglycemia.     

Effect of acute hypoglycemia on human cerebral glucose metabolism measured by ¹³C magnetic resonance spectroscopy

OBJECTIVE

To investigate the effect of acute insulin-induced hypoglycemia on cerebral glucose metabolism in healthy humans, measured by ¹³C magnetic resonance spectroscopy (MRS).

RESEARCH DESIGN AND METHODS

Hyperinsulinemic glucose clamps were performed at plasma glucose levels of 5 mmol/L (euglycemia) or 3 mmol/L (hypoglycemia) in random order in eight healthy subjects (four women) on two occasions, separated by at least 3 weeks. Enriched [1-¹³C]glucose 20% w/w was used for the clamps to maintain stable plasma glucose labeling. The levels of the ¹³C-labeled glucose metabolites glutamate C4 and C3 were measured over time in the occipital cortex during the clamp by continuous ¹³C MRS in a 3T magnetic resonance scanner. Time courses of glutamate C4 and C3 labeling were fitted using a one-compartment model to calculate metabolic rates in the brain.

RESULTS

Plasma glucose ¹³C isotopic enrichment was stable at 35.1 ± 1.8% during euglycemia and at 30.2 ± 5.5% during hypoglycemia. Hypoglycemia stimulated release of counterregulatory hormones (all P < 0.05) and tended to increase plasma lactate levels (P = 0.07). After correction for the ambient ¹³C enrichment values, label incorporation into glucose metabolites was virtually identical under both glycemic conditions. Calculated tricarboxylic acid cycle rates (V_TCA) were 0.48 ± 0.03 μmol/g/min during euglycemia and 0.43 ± 0.08 μmol/g/min during hypoglycemia (P = 0.42).

CONCLUSIONS

These results indicate that acute moderate hypoglycemia does not affect fluxes through the main pathways of glucose metabolism in the brain of healthy nondiabetic subjects.Hypoglycemia is a major threat for brain function because the brain depends on a continuous glucose supply as principal source of energy. Thus, glucose counterregulatory responses are usually initiated when glucose levels fall below ∼3.8 mmol/L to quickly restore euglycemia and maintain sufficient glucose delivery to the brain (1). The glucose level at which cognitive function declines is not fixed but depends on the complexity of the cognitive task and the cognitive domain that is tested. Nevertheless, although simple motor functions may be sustained despite even quite severe degrees of hypoglycemia, many aspects of cognitive performance become impaired at glucose levels between 3.1 and 3.4 mmol/L (2). During complex cognitive tasks, such as with motor vehicle driving, deterioration can already be observed at glucose levels as high as ∼3.8 mmol/L (3).Although the importance of maintaining sufficient glucose supply to the brain has been known for long, it is still unclear how hypoglycemia affects subsequent cerebral glucose metabolism. Various studies have indicated altered cerebral glucose handling during even mild symptomatic hypoglycemia. When the brain is supplied with an alternative energy source during hypoglycemia, such as lactate, the threshold level for initiation of glucose counterregulation shifts to lower glucose levels (4) and performance on cognitive function tests is maintained better (5). In accordance, upregulation of lactate transport into the brain during hypoglycemia has been associated with glucose counterregulatory defects (6). Using ¹H magnetic resonance spectroscopy (MRS), Bischof et al. (7) reported discrete effects of moderate hypoglycemia (∼3.1 mmol/L glucose) on cerebral glucose-derived metabolite levels in healthy volunteers. Finally, positron emission tomography (PET) studies with fluor-18-fluorodeoxyglucose (FDG) and [¹¹C]-O-methyl-d-glucose (CMG) have demonstrated regional, but not global, changes in cerebral glucose metabolism based on tracer uptake in the brain during hypoglycemia in patients with diabetes (8,9). Thus, many reports suggest that human brain glucose metabolism changes under hypoglycemic conditions, but the exact changes are unclear (10). In addition, neither with ¹H MRS nor with PET can the cerebral metabolic rate of glucose conversion into its metabolites be determined.With ¹³C MRS, it is possible to study the dynamics of glucose metabolism in vivo in the human brain. Because the natural abundance of ¹³C is only 1.1%, it can be applied as a nonradioactive magnetic resonance tracer. For this purpose, often ¹³C enriched glucose labeled at the C-1 position is used (11). With this method, the uptake of glucose in brain tissue, as well as its conversion into several downstream metabolites, can be followed over time. To optimize the intensity of the ¹³C signals of these metabolites, which occur at rather low concentration, most studies applying dynamic ¹³C MRS to the human brain have been performed under hyperglycemic conditions. We previously developed a specific protocol that has enabled us to apply ¹³C MRS with infusion of ¹³C-labeled glucose under both euglycemic and hypoglycemic conditions in human volunteers (12). This allows for mathematical modeling and calculation of metabolic fluxes of glucose metabolism (13,14). The aim of the current study was to compare human in vivo brain glucose metabolism under euglycemic and hypoglycemic conditions using ¹³C MRS.     

Activation of ATP-sensitive K+ channels in the ventromedial hypothalamus amplifies counterregulatory hormone responses to hypoglycemia in normal and recurrently hypoglycemic rats

The mechanism(s) by which glucosensing neurons detect fluctuations in glucose remains largely unknown. In the pancreatic beta-cell, ATP-sensitive K+ channels (K ATP channels) play a key role in glucosensing by providing a link between neuronal metabolism and membrane potential. The present study was designed to determine in vivo whether the pharmacological opening of ventromedial hypothalamic K ATP channels during systemic hypoglycemia would amplify hormonal counterregulatory responses in normal rats and those with defective counterregulation arising from prior recurrent hypoglycemia. Controlled hypoglycemia (approximately 2.8 mmol/l) was induced in vivo using a hyperinsulinemic (20 mU x kg(-1) x min(-1)) glucose clamp technique in unrestrained, overnight-fasted, chronically catheterized Sprague-Dawley rats. Immediately before the induction of hypoglycemia, the rats received bilateral ventromedial hypothalamic microinjections of either the potassium channel openers (KCOs) diazoxide and NN414 or their respective controls. In normal rats, both KCOs amplified epinephrine and glucagon counterregulatory responses to hypoglycemia. Moreover, diazoxide also amplified the counterregulatory responses in a rat model of defective hormonal counterregulation. Taken together, our data suggest that the K ATP channel plays a key role in vivo within glucosensing neurons in the ventromedial hypothalamus in the detection of incipient hypoglycemia and the initiation of protective counterregulatory responses. We also conclude that KCOs may offer a future potential therapeutic option for individuals with insulin-treated diabetes who develop defective counterregulation.     

Diabetes impairs hypothalamo-pituitary-adrenal (HPA) responses to hypoglycemia,and insulin treatment normalizes HPA but not epinephrine responses

We recently established that in addition to plasma adrenocorticotrophic hormone (ACTH) and corticosterone, hypothalamic corticotrophin-releasing hormone (CRH) mRNA and hippocampal type 1 glucocorticoid receptor (GR1) mRNA were also upregulated in uncontrolled streptozotocin-induced diabetes. In the current study, control, diabetic, and insulin-treated diabetic rats underwent a hyperinsulinemic-hypoglycemic glucose clamp to evaluate central mechanisms of hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia. Increases in plasma ACTH, corticosterone, and epinephrine were significantly lower in diabetic rats versus controls. Insulin treatment restored ACTH and corticosterone but not epinephrine responses to hypoglycemia in diabetic rats. Glucagon and norepinephrine responses to hypoglycemia were not affected by diabetes or insulin treatment. In response to hypoglycemia, hypothalamic CRH mRNA and pituitary proopiomelanocortin mRNA expression increased in control and insulin-treated but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal GR1 mRNA expression in control and insulin-treated diabetic rats but not in diabetic rats. In contrast, type 2 glucocortoid receptor (GR2) mRNA was not altered by hypoglycemia. In conclusion, despite increased basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that the defect in CRH response could be related to the defective GR1 response. It is intriguing that insulin treatment restored the HPA response to hypoglycemia but, surprisingly, not the deficient epinephrine response. This is important because during severe hypoglycemia, epinephrine is an important counterregulatory hormone.     

Increased GABAergic output in the ventromedial hypothalamus contributes to impaired hypoglycemic counterregulation in diabetic rats

OBJECTIVE

Impaired glucose counterregulation during hypoglycemia is well documented in patients with type 1 diabetes; however, the molecular mechanisms underlying this defect remain uncertain. We reported that the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in a crucial glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), plays an important role in modulating the magnitude of the glucagon and epinephrine responses to hypoglycemia and investigated whether VMH GABAergic tone is altered in diabetes and therefore might contribute to defective counterregulatory responses.

RESEARCH DESIGN AND METHODS

We used immunoblots to measure GAD₆₅ protein (a rate-limiting enzyme in GABA synthesis) and microdialysis to measure extracellular GABA levels in the VMH of two diabetic rat models, the diabetic BB rat and the streptozotocin (STZ)-induced diabetic rat, and compared them with nondiabetic controls.

RESULTS

Both diabetic rat models exhibited an ~50% increase in GAD₆₅ protein as well as a twofold increase in VMH GABA levels compared with controls under baseline conditions. Moreover, during hypoglycemia, VMH GABA levels did not change in the diabetic animals, whereas they significantly declined in nondiabetic animals. As expected, glucagon responses were absent and epinephrine responses were attenuated in diabetic rats compared with their nondiabetic control counterparts. The defective counterregulatory response in STZ-diabetic animals was restored to normal with either local blockade of GABA_A receptors or knockdown of GAD₆₅ in the VMH.

CONCLUSIONS

These data suggest that increased VMH GABAergic inhibition is an important contributor to the absent glucagon response to hypoglycemia and the development of counterregulatory failure in type 1 diabetes.Iatrogenic severe hypoglycemia is the most serious acute complication in insulin-treated diabetes, and it remains the limiting factor in maintaining proper glycemic control (1,2). The brain, and particularly the ventromedial hypothalamus (VMH), plays a crucial role in sensing hypoglycemia and initiating the physiological counterregulatory responses that rapidly correct it (3–6), namely the release of glucagon and epinpehrine (7). In longstanding type 1 diabetes, however, these mechanisms are either lost or become impaired, making these individuals more susceptible to the threat of hypoglycemia (8). Although the mechanism(s) underlying these defects have not been identified, it has been postulated that impaired glucose counterregulation in type 1 diabetes stems from a number of factors. The first of which is the simultaneous loss of endogenous insulin secretion and, in association, the capacity to release glucagon in response to hypoglycemia (9). The latter is thought to be the result of a number of intra- and extrapancreatic factors, including the loss of β-cells and thus the capacity to suppress the local release of insulin (10–12), zinc (13,14), and the neurotransmitter γ-aminobutyric acid (GABA) (15) during hypoglycemia. These factors together, when coupled with excessive administration of exogenous insulin, act to suppress glucagon release. Second, adaptations that occur within the central and peripheral nervous system have been implicated in the impaired glucagon as well as epinephrine responses as well, including alterations in brain glucose and monocarboxylic acid transport and metabolism, and changes in neural innervation of the islet (16–23). The precise mechanisms and the relative contributions of the many disturbances in peripheral and central signals to counterregulatory failure in type 1 diabetes still are unclear.Glucose and glucose deprivation have been shown to alter GABA levels within the brain (24–27), but the evidence for its role in regulating glucose counterregulation remains somewhat controversial. Our laboratory reported that GABA acts within the VMH to modulate the magnitude of both the glucagon and epinephrine responses to hypoglycemia in nondiabetic rats (28). Subsequently, we demonstrated that increased GABAergic tone in the VMH was an important contributor to counterregulatory failure in nondiabetic rats exposed to recurrent antecedent hypoglycemia (29). Studies in nondiabetic humans also have shown that activation of GABA_A receptors with systemic delivery of the benzodiazepine analog, alprazolam, reduces counterregulatory and neuroendocrine responses to hypoglycemia in primates and healthy human subjects (30–32). Conversely, administration of modafinil to healthy human subjects to lower brain GABA concentrations was reported to improve adrenergic sensitivity and some aspects of cognitive function during hypoglycemia but did not significantly affect counterregulatory hormone release (33).The current study was undertaken to investigate whether GABA inhibitory tone is increased in the VMH in two rodent models of type 1 diabetes and whether GABA contributes to defective counterregulation during hypoglycemia in diabetes. Our data suggest, for the first time, that increased hypothalamic GABAergic neurotransmission plays a significant role in the loss of the glucagon response as well as the impairment of epinephrine release seen in rats with type 1 diabetes during acute hypoglycemia and that these counterregulatory defects are reversed by specifically reducing excessive GABA tone in the VMH.     

Effects of Intensive Therapy and Antecedent Hypoglycemia on Counterregulatory Responses to Hypoglycemia in Type 2 Diabetes

OBJECTIVE—The physiology of counterregulatory responses during hypoglycemia in intensively treated type 2 diabetic subjects is largely unknown. Therefore, the specific aims of the study tested the hypothesis that 1) 6 months of intensive therapy to lower A1C <7.0% would blunt autonomic nervous system (ANS) responses to hypoglycemia, and 2) antecedent hypoglycemia will result in counterregulatory failure during subsequent hypoglycemia in patients with suboptimal and good glycemic control.RESEARCH DESIGN AND METHODS—Fifteen type 2 diabetic patients (8 men/7 women) underwent 6-month combination therapy of metformin, glipizide XL, and acarbose to lower A1C to 6.7% and 2-day repeated hypoglycemic clamp studies before and after intensive therapy. A control group of eight nondiabetic subjects participated in a single 2-day repeated hypoglycemic clamp study.RESULTS—Six-month therapy reduced A1C from 10.2 ± 0.5 to 6.7 ± 0.3%. Rates of hypoglycemia increased to 3.2 episodes per patient/month by study end. Hypoglycemia (3.3 ± 0.1 mmol/l) and insulinemia (1,722 ± 198 pmol/l) were similar during all clamp studies. Intensive therapy reduced (P < 0.05) ANS and metabolic counterregulatory responses during hypoglycemia. Antecedent hypoglycemia produced widespread blunting (P < 0.05) of neuroendocrine, ANS, and metabolic counterregulatory responses during subsequent hypoglycemia before and after intensive therapy in type 2 diabetic patients and in nondiabetic control subjects.CONCLUSIONS—Intensive oral combination therapy and antecedent hypoglycemia both blunt physiological defenses against subsequent hypoglycemia in type 2 diabetes. Prior hypoglycemia of only 3.3 ± 0.1 mmol/l can result in counterregulatory failure in type 2 diabetic patients with suboptimal control and can further impair physiological defenses against hypoglycemia in intensively treated type 2 diabetes.Large randomized controlled multicenter clinical trials have demonstrated the benefit of improved glycemic control on microvascular complications in both type 1 and type 2 diabetes (1,2). These compelling data have produced a paradigm shift in the treatment of diabetes (particularly type 2 diabetes) striving for A1C values <7.0% (3). The major drawbacks of tight metabolic control in patients with type 1 diabetes are well documented and include increased hypoglycemia and weight gain (4–8).Recently, three large studies have investigated the effects of rigorous metabolic control (A1C <7.0%) on the prevalence of macrovascular disease in type 2 diabetes (9–11). The overall conclusion of these studies was that A1C values <7.0% did not produce a statistically significant reduction in macrovascular events but did produce a marked increase in hypoglycemia in type 2 diabetes. The effects of intensive therapy on physiological counterregulatory responses during hypoglycemia in type 2 diabetes have not been thoroughly investigated. Burge et al. (12) demonstrated that improving glycemic control during an 8-day in-patient admission could lower symptom responses and plasma glucose levels for activation of epinephrine during hypoglycemia. Levy et al. (13), using a cross-sectional study design, also concluded that improved glycemic control in type 2 diabetes shifts the thresholds for counterregulatory hormone release to lower plasma glucose concentrations during hypoglycemia. Korzon-Burakowska et al. (14) improved A1C from 11.3 ± 1.1 to 8.1 ± 0.9% during a 4-month period. Thresholds (i.e., plasma glucose values) for counterregulatory hormone release and epinephrine and cortisol responses were lowered by improved glycemic control. Spyer et al. (15), investigating a group of seven type 2 diabetic patients with an A1C of 7.4%, also found that the glycemic thresholds for counterregulatory hormone release were reduced from elevated to normal physiological glucose levels. However, similar to some (13,16) but not all studies (17), there was no difference in values of the key counterregulatory hormones, epinephrine and glucagon, during hypoglycemia when compared with nondiabetic control subjects. Studies investigating the mechanisms regulating counterregulatory responses during hypoglycemia in type 2 diabetes are even fewer. Segel et al. (16) determined that antecedent hypoglycemia in a group of type 2 diabetic patients with an A1C of 8.1% resulted in hypoglycemia-associated autonomic failure similar to patients with type 1 diabetes. Despite the above data, two questions remain unanswered: 1) What are the effects of a period of rigorous glycemic control to reduce A1C <7.0% on counterregulatory responses in type 2 diabetes, and 2) what are the effects of antecedent hypoglycemia on autonomic nervous system (ANS), neuroendocrine, and metabolic counterregulatory mechanisms before and after a period of rigorous metabolic control in type 2 diabetes? In the present study, we tested the hypothesis that 6-month intensive therapy to lower A1C <7.0% would impair counterregulatory response to hypoglycemia and that antecedent hypoglycemia would further impair key homeostatic counterregulatory mechanisms during subsequent hypoglycemia in type 2 diabetes.     

Further defects in counterregulatory responses induced by recurrent hypoglycemia in IDDM.

We evaluated the effect of previous experimental hypoglycemia on counterregulatory responses to hypoglycemia in 13 IDDM patients. These patients had defects in counterregulatory responses to hypoglycemia compared with 7 nondiabetic control subjects. Plasma EPI and glucagon responses to hypoglycemia in IDDM patients were approximately 60% of levels in nondiabetic subjects (P less than 0.02 and P less than 0.001, respectively). Hepatic glucose output ([3-3H]glucose) was reduced by approximately 60% of normal (P less than 0.005), and the glucose infusion rate required to maintain plasma glucose was correspondingly greater in people with IDDM (P less than 0.001). With a modified glucose clamp (plasma insulin approximately 330 pM), the diabetic subjects underwent two sequential 120-min periods of hypoglycemia (approximately 3.0 mM) with an intervening 60-min euglycemic recovery period. In the IDDM patients, there were 30-50% decreases in plasma GH (P less than 0.005) and cortisol (P less than 0.001) responses during the second hypoglycemic period compared with the first. In addition, glucose output, already defective compared with that in nondiabetic subjects, was further reduced by 33% (P = 0.03) during the second period of experimental hypoglycemia. There was no effect of repeated hypoglycemia on the responses of plasma glucagon, EPI, or NE, though plasma EPI was correlated directly with glucose output (P less than 0.001) and inversely with glucose uptake (P less than 0.05). There was no correlation between the rise in glucose output during hypoglycemia and antecedent glycemic control as measured by HbA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain activation during working memory is altered in patients with type 1 diabetes during hypoglycemia

OBJECTIVE

To investigate the effects of acute hypoglycemia on working memory and brain function in patients with type 1 diabetes.

RESEARCH DESIGN AND METHODS

Using blood oxygen level–dependent (BOLD) functional magnetic resonance imaging during euglycemic (5.0 mmol/L) and hypoglycemic (2.8 mmol/L) hyperinsulinemic clamps, we compared brain activation response to a working-memory task (WMT) in type 1 diabetic subjects (n = 16) with that in age-matched nondiabetic control subjects (n = 16). Behavioral performance was assessed by percent correct responses.

RESULTS

During euglycemia, the WMT activated the bilateral frontal and parietal cortices, insula, thalamus, and cerebellum in both groups. During hypoglycemia, activation decreased in both groups but remained 80% larger in type 1 diabetic versus control subjects (P < 0.05). In type 1 diabetic subjects, higher HbA_1c was associated with lower activation in the right parahippocampal gyrus and amygdala (R² = 0.45, P < 0.002). Deactivation of the default-mode network (DMN) also was seen in both groups during euglycemia. However, during hypoglycemia, type 1 diabetic patients deactivated the DMN 70% less than control subjects (P < 0.05). Behavioral performance did not differ between glycemic conditions or groups.

CONCLUSIONS

BOLD activation was increased and deactivation was decreased in type 1 diabetic versus control subjects during hypoglycemia. This higher level of brain activation required by type 1 diabetic subjects to attain the same level of cognitive performance as control subjects suggests reduced cerebral efficiency in type 1 diabetes.Acute episodes of hypoglycemia are a rate-limiting adverse effect in the treatment of type 1 diabetes. When severe, they can lead to seizures and coma (1). Even mild to moderate hypoglycemia is known to impair cognitive functions, such as working memory (2,3). Working memory is used to actively maintain and manipulate information over a brief period of time and to allocate attentional resources among competing subtasks (4,5). Traditionally, working-memory performance is thought to depend primarily on a network of brain regions, including portions of the frontal and parietal lobes, thalamus, precuneus, cerebellum, and insula (6,7).Using blood oxygen level–dependent (BOLD) functional magnetic resonance imaging (fMRI), we evaluated how diabetes impacts these neural processes under euglycemic and hypoglycemic conditions when subjects were presented with a working-memory task (WMT). Diabetes is known to negatively affect working memory (8). This task evaluates functional effects that might reflect changes in brain structure and/or presage decreases in cognitive performance. A better understanding of the brain’s metabolic and physiological mechanisms underlying the cognitive functions implicated in working memory could lead to improved treatment strategies to help maintain cortical function in patients with diabetes during hypoglycemia (9).BOLD fMRI is a well-established method for examining regional brain activation in response to physiological, pharmacological, sensory, or cognitive tasks (10). Studies that have examined brain activation in response to sensory stimulation or cognitive challenges using BOLD fMRI during hypoglycemic conditions in nondiabetic subjects (11–13) have shown that hypoglycemia reduces regional brain BOLD activation. This reduction in BOLD response during hypoglycemia has been attributed to low glucose levels causing decreases in neuronal activity, glucose oxidative metabolism, cerebral blood flow, neurovascular coupling, and/or neuronal recruitment (12).Whether cognitive function in patients with type 1 diabetes is affected by hypoglycemia in the same manner as in nondiabetic individuals remains unclear because few studies using functional neural imaging have directly compared diabetic and nondiabetic subjects during the performance of cognitive tasks (14,15). If brain glucose transport or metabolism are altered in type 1 diabetes, as has been suggested in recent studies by our group (16) and others (17), then one would expect that the BOLD activation response during hypoglycemia may differ between diabetic patients compared with nondiabetic control subjects. On the basis of these findings, we hypothesized that 1) patients with type 1 diabetes would have greater BOLD activation during the performance of a WMT during hypoglycemia when compared with nondiabetic control subjects, 2) cognitive performance would deteriorate during hypoglycemia in both groups, and 3) among type 1 diabetic patients, better glycemic control (lower HbA_1c) would correlate with BOLD activation responses to the WMT during hypoglycemia. We also conducted exploratory analyses to examine deactivation patterns in the default-mode network (DMN), the regions of the brain that are more active during rest (18), because of other research by our group examining the effects of diabetes on deactivation patterns during cognitive tasks and previous research suggesting that DMN function may be altered in diseases that affect cognition, such as Alzheimer’s disease (19).