1 Program in Neuroscience, University of Illinois at Urbana-Champaign, Urbana, Illinois
2 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois
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ABSTRACT |
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Hypoglycemia initiates compensatory responses, most notably the release of glucagon from the endocrine pancreas and sympathoadrenal activation resulting in secretion of epinephrine. There is strong evidence that the brain, in particular the hypothalamus, plays an important role in autonomic control of glucose balance (rev. in 1). Detection of local glucoprivation in the brain may be critical to the generation of appropriate compensatory responses (2). The ventromedial hypothalamus (VMH) has received attention because neurons in this area are responsive to both systemic hypoglycemia (3) and to changes in local extracellular glucose concentrations (46). Local glucoprivation by application of the glucose-analogue 2-deoxyglucose into this area replicated the increased neuroendocrine and sympathoadrenal output characteristic of systemic hypoglycemia (7). In addition, compensatory responses to systemic hypoglycemia were inhibited when the VMH was bilaterally perfused with glucose (8). In vitro studies, using tissue slices, demonstrated that 30% of neurons throughout the VMH alter their firing rate to changes in glucose concentration in the bathing solution (46,9). It has been suggested that the cellular mechanisms by which hypothalamic neurons respond to glucose are similar to those of pancreatic ß-cells, in part because they involve membrane-bound sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels (4,6,9). Neurons in the VMH of mice lacking Kir6.2, one of the subunits of KATP channels, are less responsive to changes in ambient glucose (10).
To be able to function as glucose sensors to initiate compensatory responses, neurons must be sensitive to physiological changes in ambient glucose levels. As a wide range of glucose values have been reported, it remains unclear what glucose levels are in the brains of intact animals. Glucose concentrations in brain interstitial fluid under euglycemic conditions have been examined with the zero-net-flux method using in vivo microdialysis, first described by Lönnroth et al. (11). They were found to be 0.30.5 mmol/l in striatum (12,13), 1 mmol/l in hippocampus (14), and 3.3 mmol/l in the neocortex (15). Other reports, using a microelectrode coated with glucose-oxidase (16), indicated 0.351 mmol/l in striatum (17) and 2.4 mmol/l in cingulate cortex (18). To our knowledge, interstitial glucose levels in the hypothalamus have not yet been reported, nor are there reports of changes in hypothalamic glucose during systemic hypoglycemia. Bequet et al. (19) reported that during prolonged insulin-induced hypoglycemia, or after a 36-h fast, extracellular glucose in the cortex was closely coupled to glucose in the blood. Glucose is likely taken into the brain by facilitated diffusion; however, controversy remains as to what is the rate-limiting factor for the supply of glucose to the hypothalamus. Under normal conditions, glucose supply from the blood is believed to be in excess of metabolic requirements (2022). On the other hand, increased neural activity may also influence the local extracellular glucose concentration (12,23).
Intensive insulin therapy is used in the treatment of type 1 diabetes to improve metabolic control and to reduce long-term diabetes complications. Unfortunately, frequent iatrogenic antecedent hypoglycemia diminishes warning symptoms and endogenous compensatory responses to subsequent hypoglycemia (24,25). This syndrome of "hypoglycemia unawareness" is responsible for considerable physical and psychological morbidity in diabetes (rev. in 26). It has been hypothesized that hypoglycemia unawareness is mediated by an increased efficiency of brain glucose uptake (27). While preserving the brains glucose supply, detection of systemic hypoglycemia by putative brain glucose sensors may be impaired. There is some experimental evidence to support this theory. Lower symptom scores and blunted hormonal responses in type 1 diabetic patients with good glycemic control are associated with preserved brain glucose uptake and protected neural function during hypoglycemia (28). Similar findings have been reported in rats, where chronic hypoglycemia inhibited peripheral catecholamine secretion, increased glucose extraction by the brain (29,30), and even protected brainstem function (31). In the present study, the first objective was to determine, in vivo, the interstitial concentration of glucose in the VMH during systemic euglycemia and during an acute episode of hypoglycemia. We expected interstitial glucose concentrations in the VMH would decrease proportionally to a decrease in blood glucose. The second objective of this study was to evaluate the effect of recurrent hypoglycemia on VMH glucose and test the hypothesis that after repeated episodes of antecedent hypoglycemia there is less change in interstitial glucose concentrations during systemic hypoglycemia.
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RESEARCH DESIGN AND METHODS |
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Surgical procedures.
After a 1-week acclimation period, rats were fitted with a jugular vein catheter and microdialysis guide cannula as previously described (3). Briefly, rats were anesthetized with a mixture of Ketamine HCl, Xylazine HCl, and Acepromazine (30:6:1 mg/kg i.m.) and a 4-cm segment of Silastic tubing (0.64 mm i.d. x 0.94 mm o.d.) was inserted into the isolated right jugular vein. The catheter was exteriorized through an incision on top of the head, a piece of 21 gauge stainless steel tubing was inserted onto the end of the catheter, and the catheter was filled with a 40% polyvinylpyrrolidone (PVP) solution containing 500 units ml heparin and capped with a sealed piece of Tygon tubing to maintain patency. The rat was placed into a stereotaxic instrument (ASI instruments, Warren, MI), and a guide cannula was positioned 2 mm dorsal to the left VMH using the stereotaxic atlas of Paxinos and Watson (32). Coordinates for the tip of the guide cannula were AP = -2.3, L = 0.8, and D = 6.4 mm relative to Bregma. The tips of the microdialysis probes were designed to extend 2 mm beyond the end of the guide to direct the surface of the membrane into the lateral edge of the anterior portion of the VMH, immediately anterior to the perifornical area. Location of cannula placement is depicted in Fig. 1. The guide cannula and the end of the venous catheter were fixed in position with dental acrylic cement and anchored to the skull with four stainless-steel screws (Small Parts, Miami Lakes, FL). After surgery, rats were monitored until they had completely recovered from the anesthesia. Postsurgical analgesia was provided with Banamine (1.5 mg/kg s.c.). At the end of the study, cannula placement was verified histologically.
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Experiment 1: Zero-net-flux experiment to determine extracellular glucose concentration in the VMH under baseline conditions, both in fed and overnight-fasted rats.
Rats were placed into one of two groups and had either free access to food (n = 11) or food was removed at the beginning of the dark period before the day of the experiment (n = 10). The zero-net-flux method with regression analysis, adapted from Lönnroth et al. (11), was used to calculate the glucose concentration in the interstitial space. In each animal, the probe was perfused with a series of Krebs Ringer buffers containing 0, 0.5, 1, or 1.5 mmol/l glucose in order of increasing concentration. At each level, the glucose flux across the probes membrane was allowed to equilibrate for 20 min. A dialysate sample was then collected for 10 min. The net glucose flux (out in) was measured at every perfusate concentration and regression analysis used to calculate the actual concentration of interstitial glucose in the VMH. Blood samples were collected at the midpoint of each 10-min sample period from the jugular vein catheter.
Experiment 2: Effect of different degrees of acute hypoglycemia on extracellular glucose in the VMH of fed rats.
Glucose concentrations in blood and extracellular fluid of the VMH were evaluated to a single intravenous administration of insulin (0.5 or 5.0 units kg/ml; Humulin, Eli Lilly, Indianapolis, IN) or an equal volume of saline. Insulin or saline was injected via the jugular vein catheter at t = 0 min. Blood glucose was measured at t = -10, 0, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, and 90 min, as described above. Dialysate samples for evaluation of changes in extracellular glucose were collected at 10-min intervals beginning 30 min before inducing systemic hypoglycemia.
Experiment 3: Two studies to evaluate the effect of recurrent hypoglycemia on interstitial glucose in the VMH.
In the first study, rats received a once-daily intravenous administration of saline (n = 6) or insulin (1.0 units kg/ml, n = 9) for 3 consecutive days. The injections were given in the middle of the light period. At the end of the light period on day 3, food was removed and all animals were fasted overnight. On day 4, blood and interstitial glucose were evaluated before and after an intravenous injection of 0.5 units/kg insulin. All rats received insulin. Timing and sample collection were as described in experiment 2.
The zero-net-flux procedure was used in a second study to confirm the changes in basal glucose levels observed in the first study. To remove the possible confounding effect of fasting, rats in this study were allowed ad libitum access to food at all times, except during the dialysis procedure. Steady-state interstitial glucose concentrations were measured after 3 consecutive days of saline (i.e., before either the first episode of hypoglycemia; n = 10) or 3 consecutive days of 1.0 units kg/ml insulin (e.g., before the fourth episode of hypoglycemia; n = 10).
Analytical methods.
Glucose in dialysate was measured with a Turner TD-700 fluorometer (Turner Designs, Sunnyvale, CA) equipped with a minicell, using a method similar to McNay et al. (14). Briefly, glucose is converted into 6-phosphate-gluconolactone in enzymatic reactions catalyzed by hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Simultaneous conversion of NADP+ to NADPH was detected fluorometrically. Dialysate samples (10 µl) were mixed with 65 µl reagent mix (in Tris buffer with pH = 8.1; 1 mmol/l MgCl2, 3 mmol/l ATP, 3 mmol/l NADP+, 3 mmol/l Dithiothreitol, 0.4 units/ml glucose-6-phosphate dehydrogenase, and 4 units/ml hexokinase) and assayed for glucose after a 60-min incubation. Filters restricted excitation wavelengths to between 300 and 400 nm and emission wavelengths to between 410 and 610 nm. The procedure was modified to provide sensitivity (threshold detection was 50 pmol glucose) to detect small changes in glucose concentration. A standard curve was linear between 0 and 250 µmol/l, and samples were adjusted to fall into this range by dilution with Krebs-Ringer bicarbonate. Inter- and intra-assay variation was between 2 and 5%. Blood glucose measurements were obtained using an Accu-Chek Instant (Roche Diagnostics, Indianapolis, IN) for experiment 2 and the first part of experiment 3. A Beckman Glucose Analyzer 2 (Beckman Instruments) was used in the zero-net-flux studies after separation of plasma by centrifugation.
Data analysis.
Results are presented as means ± SE. The sum change in glucose concentrations was determined using the trapezoid rule to calculate the area under the curve (AUC). Differences in baseline values between fed and fasted rats and in recurrent hypoglycemic episodes were analyzed by Students t test. Differences among treatment groups in responses of blood glucose, extracellular glucose, and brain/blood ratios over time were analyzed by repeated-measures ANOVA, and AUC was evaluated by one-way ANOVA. When ANOVAs were significant, a Scheffés multiple-comparison test was used to determine differences among treatment groups. Changes from baseline were analyzed by repeated-measures ANOVA followed by paired t tests. In vitro recoveries, determined using an unstirred 1 mmol/l glucose solution, were 4.6 ± 0.8%.
Supplies.
Ketamine, Acepromazine, and Butorphenol were obtained from Aveco (Fort Dodge, IA). Xylazine was obtained from Vedco (St. Joseph, MO). All other reagents were purchased from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Experiment 3.
There was no difference in the degree or pattern of hypoglycemia induced after the first or fourth dose of insulin (Fig. 3, top). Similar to the results in the second experiment, the decline (F9,90 = 27.37, P < 0.0001) in interstitial glucose concentrations in the VMH paralleled the changes in blood glucose (Fig. 3, middle). The lowest levels were reached during the 20- to 30-min interval, 0.22 ± 0.05 and 0.08 ± 0.02 mmol/l in the first and fourth hypoglycemic episodes, respectively. Throughout the fourth hypoglycemic episode, VMH glucose values remained lower than during the first hypoglycemic episode with total decrease in VMH glucose, as determined by AUC; this was the same for both groups (P = 0.89). Surprisingly, baseline glucose concentrations in the VMH were lower after 3 days of insulin treatment (0.79 ± 0.10 vs. 0.60 ± 0.10). The baseline glucose values before the first episode were similar to those of fasted rats in the first zero-net-flux study. This decrease was confirmed in the second zero-net-flux study in which rats were not fasted overnight before testing (Table 2).
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DISCUSSION |
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A relatively stable relationship between interstitial glucose concentration in the VMH and blood glucose apparent in control rats was only observed during a moderate degree of insulin-induced hypoglycemia. In these situations VMH glucose changed proportionally with the decline in blood glucose, remaining at 20% of blood values, consistent with previous reports of extracellular glucose changing in parallel with changes in blood glucose (18,19). The stable VMH-to-blood ratio during moderate hypoglycemia is suggestive of a constant, predictable relationship between the methods for determining glucose concentrations in the different fluids. The higher VMH-to-blood ratio during the first 10-min sample period is consistent with blood glucose levels falling faster than the fall in interstitial glucose and may reflect a limited buffering capacity. Any buffering capacity was quickly overwhelmed when a more severe degree of systemic hypoglycemia was induced. VMH glucose concentrations declined to a greater degree than blood glucose, to
10% of blood glucose levels. The steady decline in this ratio would be expected if glucose transport into the VMH was reduced, perhaps by a decrease in glucose uptake, a decrease in blood flow to the VMH, or if glucose metabolism in VMH was increased to a greater degree than glucose uptake from the blood. We are unaware of any reports documenting reduced blood-to-brain glucose transport or reduced blood flow in the hypothalamus during hypoglycemia. To the contrary, both cerebral blood flow (27,34) and glucose extraction (29,35) were reported to be increased during hypoglycemia. A decline in interstitial glucose may be related to increased metabolic and neural activity in the VMH, as ambient interstitial glucose was reduced when neural activity was induced (12,18,36). During hypoglycemia there is an increase in noradrenergic and GABAergic activity in the VMH (3).
The lower steady-state interstitial glucose concentrations in the VMH following repeated episodes of hypoglycemia are difficult to reconcile with reports of an increase (29,35) or of no change (37) in brain glucose uptake and the suggestion that hypoglycemia unawareness is the result of enhanced brain glucose uptake (38). Receptor density of GLUT1 (localized on capillary endothelium of the blood-brain barrier) was increased after 1 week of chronic hypoglycemia (3942). We are unaware of any studies documenting changes in GLUT1 expression after a single episode or multiple separate episodes of hypoglycemia. An increase in GLUT1 expression would support glucose uptake as being increased and, if true, an increase in cellular (i.e., neuronal and glial) glucose uptake and metabolism would be expected to account for the lower interstitial glucose. An increase in GLUT3, the primary isoform-associated glucose uptake into neurons, would be consistent with increased cellular uptake of glucose. However, neither an increase in GLUT3 expression nor an increase in cerebral glucose metabolism has been consistently measured during hypoglycemia (39,40). If glucose transport into the brain is increased, the lower steady-state glucose concentrations may reflect an increase in glycogen. Brain glycogen levels reduced during hypoglycemia rebounded to almost threefold the concentration before hypoglycemia (43). Fillenz et al. (36) proposed a role of glial glycogen in maintaining interstitial glucose concentrations, and the slight delay in the decline in VMH glucose could reflect mobilization of glycogen reserves from astrocytes, which contain glucose-6-phosphatase (44). A neuroprotective role for brain glycogen, which may also contribute to hypoglycemic unawareness, proposed by Fillenz et al. (36) and Choi et al. (43), is not supported by the present results. If greater glycogen was present in the VMH, the VMH-to-blood glucose ratio should have been improved during the fourth consecutive episode of hypoglycemia. In the present study, the fall in VMH glucose concentration was greater during the fourth hypoglycemic episode than during the first.
Surprisingly, a stable relationship between interstitial and blood glucose concentrations was not in evidence after a moderate chronic decrease in blood glucose (e.g., an overnight fast). Steady-state basal levels of glucose were reduced to a greater extent in the VMH than in blood, as evidenced by the reduced brain-to-blood ratio. This result contrasts that of Bequet et al. (19) who measured a similar decline (32%) in both blood and cortical glucose following a 36-h fast. Steady-state glucose levels were also lower after recurrent episodes of moderate hypoglycemia and were further reduced by fasting. This result indicates that the mechanisms responsible for the lower steady-state VMH glucose after an overnight fast or recurrent hypoglycemia may be independent. During moderate episodes of hypoglycemia, the interstitial glucose declined to a similar degree (7580% of baseline) during induced hypoglycemia regardless of steady-state levels before inducing hypoglycemia.
Ambient glucose concentrations in the VMH influence the activity of glucosensory neurons that likely initiate compensatory responses during hypoglycemia (4). Lesions of VMH suppress counterregulatory response (45), and maintaining a local glucose concentration in the VMH during systemic hypoglycemia reduced the increase in both plasma catecholamines and glucagon (8). The neurochemical phenotype of glucosensory neurons is uncertain; however, noradrenergic activity in the VMH was increased during an episode of induced hypoglycemia (3) and noradrenergic circuits in the VMH affect circulating concentrations of glucose and glucose-mobilizing hormones (46). Stimulation of VMH increased sympathetic efferent activity in the liver, hepatic glucose output (47,48), metabolic rate (49), and adrenal nerve activity (50). It is unclear from the present results whether activation of compensatory responses occurs in response to a relative change in interstitial glucose or an absolute change, such as reaching a threshold level. After an overnight fast, steady-state glucose levels in the VMH were lower than the nadir reached during an induced hypoglycemic episode; however, compensatory mechanisms are fully activated only in the latter situation. Most human studies evaluating physiological responses to hypoglycemia tend to fast overnight, while rodent studies do not. The rate of decline in ambient glucose concentrations may be an important factor in altering neural activity in the VMH. Most studies induce rapid declines in available glucose that would differ from the expected gradual decline expected during an overnight fast.
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ACKNOWLEDGMENTS |
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The authors are grateful for the technical assistance of Nick Guedet and Zachery Stahlschmidt and for the valuable input from Ewan McNay and Clint Canal concerning the fluorometric glucose assay.
Address correspondence and reprint requests to J. Lee Beverly, PhD, 1207 W. Gregory Dr., Urbana, IL 61801. E-mail: beverly1{at}uiuc.edu
Received for publication May 6, 2003 and accepted in revised form August 14, 2003
AUC, area under the curve; KATP, ATP-sensitive potassium; PVP, polyvinylpyrrolidone; VMH, ventromedial hypothalamus
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REFERENCES |
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