Repeated Activation of Noradrenergic Receptors in the Ventromedial Hypothalamus Suppresses the Response to Hypoglycemia

Abstract

Activation of the adrenergic system in response to hypoglycemia is important for proper recovery from low glucose levels. However, it has been suggested that repeated adrenergic stimulation may also contribute to counterregulatory failure, but the underlying mechanisms are not known. The aim of this study was to establish whether repeated activation of noradrenergic receptors in the ventromedial hypothalamus (VMH) contributes to blunting of the counterregulatory response by enhancing local lactate production. The VMH of nondiabetic rats were infused with either artificial extracellular fluid, norepinephrine (NE), or salbutamol for 3 hours/day for 3 consecutive days before they underwent a hypoglycemic clamp with microdialysis to monitor changes in VMH lactate levels. Repeated exposure to NE or salbutamol suppressed both the glucagon and epinephrine responses to hypoglycemia compared to controls. Furthermore, antecedent NE and salbutamol treatments raised extracellular lactate levels in the VMH. To determine whether the elevated lactate levels were responsible for impairing the hormone response, we pharmacologically inhibited neuronal lactate transport in a subgroup of NE-treated rats during the clamp. Blocking neuronal lactate utilization improved the counterregulatory hormone responses in NE-treated animals, suggesting that repeated activation of VMH β2-adrenergic receptors increases local lactate levels which in turn, suppresses the counterregulatory hormone response to hypoglycemia.

hypoglycemia, ventromedial hypothalamus (VMH), norepinephrine, lactate, counterregulatory response

Hypoglycemia is a common complication of insulin-treatment in patients with type 1 diabetes (1). The most important risk factor for experiencing an episode of severe hypoglycemia is the clinical syndrome of impaired hypoglycemia awareness that is characterized by loss of hypoglycemia warning symptoms (2, 3). Antecedent hypoglycemia can also attenuate the counterregulatory hormone responses, especially the sympathoadrenal response, to subsequent bouts of hypoglycemia, leading to what has been termed hypoglycemia-associated autonomic failure (HAAF) (4, 5). Hence, recurring exposure to hypoglycemia creates a “vicious cycle” which induces hypoglycemia unawareness in part, by suppressing the sympathoadrenal response (6, 7). The underlying pathophysiological mechanisms of HAAF are not fully understood, but it has been postulated that the brain and, in particular, the ventromedial hypothalamus (VMH), may be involved (8).

Previous studies in rats showed that norepinephrine (NE) levels increase in the VMH during hypoglycemia and act on β2-adrenergic receptors to enhance the counterregulatory hormone responses to an acute bout of hypoglycemia (9-13). However, following recurring exposure to hypoglycemia, the VMH NE response to hypoglycemia remains largely intact, but is associated with an attenuated counterregulatory response (14). Hence, there appears to be a disassociation between the VMH NE response and the defective counterregulatory hormone response. This observation is significant for 2 main reasons: (1) it suggests that the defect in the counterregulatory response lies downstream of NE release; and (2) it suggests that repeated adrenergic activation could potentially contribute to the pathogenesis of HAAF. In support of the latter notion, Ramanathan and colleagues reported that adrenergic blockade during day 1 hypoglycemia prevented suppression of the epinephrine response the next day (15). Similarly, it was recently reported that repeated administration of epinephrine to healthy human subjects in the absence of hypoglycemia can also impair the counterregulatory response to hypoglycemia the following day (16). This suggests that repeated activation of adrenergic receptors during prior antecedent hypoglycemia may contribute to the development of HAAF in humans, but the mechanism by which this occurs is still not clear.

In recent years, it has come to light that NE plays an important role in regulating astrocytic metabolism and neuronal activity within the brain (17). In particular, NE enhances glycogenolysis (18) and increases lactate production (19, 20) in astrocytes through the activation of β2-adrenergic receptors (17, 20-22); whereas in neurons, it enhances neuronal activity and increases lactate transport (23-25). While the acute effects of NE on astrocytic metabolism have been studied extensively, whether repeated activation of noradrenergic receptors in the VMH chronically raises extracellular lactate concentrations and whether such an effect is linked to the pathogenesis of HAAF has not been investigated.

The relationship between VMH NE and lactate may be important in this case because elevated VMH lactate levels contribute, at least in part, to suppressing glucose counterregulatory responses in recurrently hypoglycemic rats (26). When glucose supplies are limited, the brain can use other types of fuels such as lactate to sustain neuronal activity (26, 27). A rise in VMH lactate levels and its subsequent use as an alternate fuel substrate by glucose responsive neurons can mask the neurons’ ability to detect a fall in blood glucose levels if its energy demands are met by lactate. To this end, elevated brain lactate levels have been reported in patients with type 1 diabetes and in rodent models of recurrent hypoglycemia and type 1 diabetes (12, 28, 29). Inhibiting neuronal lactate utilization in type 1 diabetic rats and recurrently hypoglycemic rats restored brain sensing mechanisms and the counterregulatory hormone response to hypoglycemia (26, 30). The mechanisms leading to the rise in VMH lactate levels following antecedent exposure to hypoglycemia are not known but may be related to repeated activation of adrenergic receptors (15, 16).

In light of the close link between NE and lactate production (20), we investigated whether repeated activation of VMH noradrenergic receptors, in the absence of peripheral hypoglycemia, increases the local production of lactate and whether this contributes to counterregulatory failure (26, 30).

Materials and Methods

For this study, male Sprague-Dawley rats (Harlan Laboratories, South Easton, MA) with an initial body weight of ~300 g were used. After arriving at the Yale Animal Resources Center, they were individually housed in cages filled with shredded paper, a hut, and gnawing block for environmental enrichment, in a temperature and humidity-controlled room. The animals had free access to water and standard rat chow (Harlan Teklab, Indianapolis, IN, USA). The animals were acclimated for 7 days before any procedures began (Fig. 1). The experimental protocols were approved by the Institutional Animal Care and Use Committee at Yale University and followed the principles of laboratory animal care.

Figure 1.

Study Time Course. Timeline illustrating acclimation period from days 0-7. Intracranial guide cannulas and vascular catheters were implanted on day 7. Daily infusion of either artificial extracellular fluid (aECF), norepinephrine (NE) or salbutamol into the VMH were performed on days 14-16 and the hypoglycemic clamp was performed on day 17 following an overnight fast.

Surgery

Following the acclimation period, the animals underwent surgery to have vascular catheters placed into the left carotid artery for blood sampling and the right jugular vein for infusion, as described previously (31). In addition, bilateral microinjection guide cannulas (PlasticsOne, Roanoke, VA, USA) that targeted the VMH were implanted (from bregma: −2.6 mm anteroposterior (AP), ±0.6 mm mediolateral (ML), and −7.6 mm dorsoventral (DV) at an angle of 0°). Following implantation of the microinjection guide cannulas, bilateral microdialysis-microinjection guide cannulas (Amuza Inc, San Diego, CA, USA) that targeted the VMH (from bregma: −2.6mm AP, ±3.8mm ML, and −8.8mm DV at angle of 16°) were also implanted. These guide cannulas were secured in place with screws and dental acrylic. Microdialysis guide cannulas were positioned to allow 1-mm microdialysis probes to target the ventrolateral portion of the VMH (32).

Recurrent Treatment Groups

Seven days after surgery, the animals were randomly assigned to one of the following 3 recurrent treatment groups:

Controls

The control animals received either an infusion of the artificial extracellular fluid vehicle (aECF [N = 3]; composition in mM: NaCl 128, KCl 3.0, CaCl₂ 1.3, MgCl₂ 1.0, NaHCO₃ 21.0, NaH₂PO₄ 1.3) (33) into the VMH for 3 hours/day for 3 consecutive days or they did not have needles inserted at all (N = 6) to control for any damage that might have arisen from repeated insertion of the microinjection needles. Artificial ECF served as the vehicle for all of the compounds used in this study.

Norepinephrine treatment

The repeated NE group received an infusion of 1 of 2 different concentrations of NE (Sigma-Aldrich, St Louis, MO, USA) into the VMH for 3 hours/day for 3 consecutive days. The lower dosage group received an infusion of 200nM NE (N = 7) whereas the higher dosage group received an infusion of 400nM NE (N = 5).

Salbutamol treatment

To determine whether the β2 subclass of adrenergic receptors was responsible for the effects of NE on VMH lactate production (17), we infused salbutamol (SAL) (200nM; Sigma-Aldrich, St. Louis, MO, USA), a short-acting β2-adrenergic receptor agonist, into the VMH of a group of rats (N = 6) for 3 hours/day for 3 consecutive days.

Compound Infusions

On each day that the compounds were infused, bilateral microinjection needles (PlasticsOne, Roanoke, VA, USA) were connected to syringes filled with the appropriate compound for infusion using polyethylene tubing. The syringes were placed into Harvard 11 Plus infusion pumps (Harvard Apparatus, Holliston, MA, USA). The microinjection needles were then inserted through the microinjection guide cannulas and extended 1 mm beyond the tip of the guide cannulas. The solutions were infused at a rate of 0.05 μL/min for 3 hours each day for 3 consecutive days. This slow infusion rate contained the injection volume within the VMH region (Supplemental Figure 1 (34)) and did not damage any neurons within the VMH (Supplemental Figure 2 (35)). During the infusion, food was withheld, and blood glucose levels were monitored at 0, 30, 60, 120, and 180 minutes from a 0.3-μl blood sample taken from a tail nick using a handheld glucometer (AlphaTRAK2, Abbott Laboratories, Chicago, IL, USA). At the end of each 3-hour infusion period, the pumps were stopped and the microinjection needles left in place for an additional 5 minutes before being removed and replaced with a dummy guide to maintain patency of the cannula. The animals were then given free access to food. On the third day, after the final treatment, the animals were given free access to food for 5 hours before being fasted overnight for the microdialysis-hypoglycemic clamp study the following day. During the overnight fast, the animals were moved into the microdialysis cages with fresh bedding to allow enough time for acclimation to the new environment.

Hypoglycemic Clamp Study With Microdialysis (Fig. 2)

The following day, the vascular catheters on the rats were connected to infusion pumps. The jugular vein was used for infusion of 20% glucose and regular insulin (Humulin R, Eli Lilly, Indianapolis, IN, USA) and the carotid artery was used for blood collection. Microdialysis-microinjection probes (1 mm; Amuza Inc, San Diego, CA, USA) were inserted through the implanted guide cannulas. The animals were then allowed to recover from handling for 2.5 to 3 hours prior to the start of microdialysate and baseline blood sample collection. Artificial ECF was constantly perfused through the microdialysis probe at a rate of 1.5 μL/min for the entire study. Following the collection of baseline blood samples, microdialysate samples were collected at 10-minute intervals for the duration of the study.

Once the baseline microdialysate samples were collected, the animals were microinjected with aECF into the VMH at a rate of 0.1 μL/min for 1 minute just prior to the start of the hypoglycemic phase of the clamp. To determine whether the NE-induced rise in VMH lactate levels contributed to counterregulatory failure, a subgroup of rats that were treated with antecedent NE infusions were microinjected with a monocarboxylic acid transporter inhibitor, alpha-4-hydroxycinnamic acid (NE+4CIN, N = 8, 15 nmol per side; Sigma-Aldrich, St. Louis, MO, USA) in place of the aECF microinjection to block neuronal lactate uptake during the hypoglycemic clamp (36).

Following microinjection, a constant insulin (50 mU/kg/min) and variable 20% dextrose infusion were initiated to lower and maintain plasma glucose levels at 45 ± 5 mg/dL for 90 minutes. Plasma glucose was assessed every 5 minutes using an Analox GM9 glucose analyzer (Analox Instruments). Blood samples were collected at 30-minute intervals throughout the clamp study for measurement of plasma glucagon and catecholamine responses. Following each blood sample collection, the red blood cells were resuspended in an equivalent volume of artificial plasma and reinfused back into the animal to prevent volume depletion and anemia (37).

At the end of the study, following collection of the final blood and microdialysate samples, the animals were euthanized with an overdose of sodium pentobarbital (Somnasol, Henry Schein Animal Health, Dublin, OH) and the brains of the animals were immediately removed and frozen on dry ice. Accuracy of probe placement was subsequently determined by inspection of coronal brain sections. Only data obtained from those animals with correctly positioned microdialysis probes were analyzed.

Hormone and Microdialysate Assays

Plasma glucagon concentrations were analyzed using commercial radioimmunoassay kits (Merck-Millipore, Darmstadt, Germany). Plasma epinephrine was measured by high-performance liquid chromatography with electrochemical detection (Thermo Scientific, Waltham, MA). VMH lactate concentrations from microdialysate samples were analyzed using the ISCUS Flex Microdialysate analyzer (M Dialysis, Johanneshov, Sweden).

Immunohistochemistry

The rats were deeply anesthetized with sodium pentobarbital and perfused with phosphate buffered saline (PBS), followed by 10% neutral buffered formalin. The brains were removed and placed into a 30% sucrose solution at 4 °C. Once sunk, the brains were coronally sectioned into 30-μm slices on a cryostat and the sections were placed into an antifreeze solution at −20 °C. Then, the sections were washed twice in PBS and transferred to a blocking solution containing 10% normal donkey serum and 0.15% Triton for 2 hours before being incubated with the primary rabbit anti-β2-adrenergic receptor antibody (1:100 dilution, cat#SAB4500577; Sigma, St Louis, MO, USA) (38) for an additional 2 hours at room temperature. Subsequently, 3% normal goat serum and either mouse anti-GFAP (glial fibrillary acid protein) (1:500 dilution, cat #G3893; Millipore-Sigma, St Louis, MO, USA) (39) or mouse anti-NeuN (neuronal marker) (1:500 dilution; cat #ab104224, Abcam, Cambridge, MA) (40) were added and incubated at 4 °C overnight. The next day, the sections were washed in PBS and incubated in fresh blocking solution containing AlexaFluor 488-conjugated donkey anti-rabbit (1:500 dilution, cat# A-21206; Thermofisher, Rockford, IL) (41) and AlexaFluor 546-conjugated goat anti-mouse (1:300 dilution; cat #A-11003; Thermofisher, Rockford, IL) (42) for 1 hour at room temperature before being counterstained with 4′,6-diamidino-2-phenylindole (DAPI, cat #D9542, Sigma-Aldrich, St. Louis, MO). Mounted tissues were then imaged using the Zeiss Axio Scan.Z1.

Evaluating Neuronal Damage

Frozen 30-μm coronal brain sections taken from animals that received intracranial implants and (1) infused repeatedly with aECF or (2) received no infusion at all, as described above, were mounted onto gelatin-coated slides and counterstained with fluoro-jade (Histo-Chem Inc., cat #1FJC) to look for neuronal damage. Mounted sections were imaged using an Olympus IX81 fluorescence microscope.

Statistical Analysis

Differences within each group were assessed by repeated-measures 2-way analysis of variance (ANOVA) or 1-way ANOVA followed by post hoc analysis using Tukey’s multiple comparisons test, as appropriate. A P value below 0.05 was considered statistically significant. Since no significant differences in microdialysate and plasma hormone responses were observed between the 2 control groups, the animals from these 2 groups were pooled together for analysis. Similarly, the low and high dose NE groups were also pooled for analysis.

Results

Plasma Glucose During the Clamp

Baseline plasma glucose concentrations were similar between treatment groups (Table 1; ANOVA P = 0.64). Mean plasma glucose concentrations during the clamp did not differ between the 4 groups (Fig. 2). Thirty minutes into the clamp, all treatment groups reached the hypoglycemic target range and this level of glycemia was maintained until 90 minutes.

Table 1.

Baseline Plasma Glucose and Counterregulatory Hormone Concentrations

	Control (n = 9)	NE (n = 12)	NE+4CIN (n = 8)	Salbutamol (n = 6)
Plasma glucose, mg/dL	109 ± 7	120 ± 7	123 ± 8	113 ± 15
Glucagon, pg/mL	82 ± 3	163 ± 61	116 ± 29	177 ± 81
Epinephrine, pg/mL	271 ± 57	399 ± 165	698 ± 205	276 ± 111

No significant differences were observed between treatment groups for any of these parameters. Data are shown as mean ± SEM.

Abbreviations: 4CIN, alpha-4-hydroxycinnamic acid; NE, norepinephrine.

Figure 2.

Study protocol and mean plasma glucose concentrations during the hypoglycemic clamp procedure. The Control group is represented by the dotted line, the norepinephrine treated group (NE) is represented by the solid line, the norepinephrine plus a lactate transporter inhibitor (NE-4CIN) is represented by the dashed line and the salbutamol group is represented by a line with triangles. Error bars depict SEM. No significant differences were noted between the 4 treatment groups.

Extracellular Lactate Levels in the VMH

Repeated administration of NE into the VMH significantly raised basal extracellular lactate concentrations in the VMH by 57% compared with controls (Fig. 3). As expected, baseline lactate levels in the NE+4CIN-treated group were similar to those in the NE-treated group. The group treated with salbutamol exhibited the highest mean lactate levels but while these concentrations were significantly elevated compared with controls, they were not significantly higher than the two NE-treated groups.

Figure 3.

Repeated activation of β2-adrenergic receptors in the VMH increases local extracellular lactate concentrations. Extracellular lactate concentrations in the VMH of Control (open diamonds = aECF Controls; closed diamonds = no needle insertion), NE-treated (open circles = high dose of NE [400nM]; closed circles = low dose of NE [200nM]), NE+4CIN-treated (open triangles = high dose of NE [400nM]; closed triangles = low dose of NE [200nM]) and salbutamol-treated (closed hexagons) rats. Treatment with either norepinephrine (NE; P < 0.01 vs Control) or salbutamol (P < 0.05 vs Control) significantly increased basal extracellular lactate concentrations compared to controls. Data presented as mean ± SEM.

The Counterregulatory Hormone Responses to Hypoglycemia

Basal plasma glucagon and epinephrine concentrations were similar between the 4 treatment groups (Table 1; ANOVA P = 0.57 and 0.24, respectively). In response to hypoglycemia, peak plasma glucagon (Fig. 4) and epinephrine (Fig. 5) responses which generally occurred between 30 and 60 minutes and between 60 and 90 minutes into the clamp, respectively, increased significantly from baseline levels in all 4 treatment groups. Repeated infusions of NE into the VMH significantly reduced peak glucagon and epinephrine responses to hypoglycemia by ~28% to 33% compared with their control counterparts (P < 0.01 and 0.05 vs controls, respectively). To determine whether the suppression induced by NE treatment was mediated through β2-adrenergic receptors, we infused salbutamol, a β2-adrenergic receptor agonist, into the VMH instead of NE. Salbutamol treatment blunted the counterregulatory hormone responses even further with both of the counterregulatory hormone responses being significantly lower than control and NE-treated rats.

Figure 4.

Repeated activation of β2-adrenergic receptors in the VMH suppresses the glucagon response to hypoglycemia. Peak plasma glucagon responses in Control (open diamonds = aECF Controls; closed diamonds = no needle insertion), NE-treated (open circles = high dose of NE [400nM]; closed circles = low dose of NE [200nM]), NE+4CIN-treated (open triangles = high dose of NE [400nM]; closed triangles = low dose of NE [200nM]) and salbutamol-treated (closed hexagons) rats presented as a percentage of the Control value. Peak responses generally occurred between 30 and 60 minutes during the hypoglycemic clamp procedure. The bars indicate the mean ± SEM for each treatment group. Both NE and salbutamol treatment significanty reduced peak glucagon responses compared to Controls (P < 0.01 and P < 0.0001, respectively). Inhibiting lactate transport in NE-treated animals using 4CIN tended to improve peak glucagon responses but the improvement did not reach statistical significance (P = 0.09 vs NE).

Figure 5.

Repeated activation of β2-adrenergic receptors in the VMH suppresses the epinephrine response to hypoglycemia. Peak plasma epinephrine responses in Control (open diamonds = aECF Controls; closed diamonds = no needle insertion), NE-treated (open circles = high dose of NE [400nM]; closed circles = low dose of NE [200nM]), NE+4CIN-treated (open triangles = high dose of NE [400nM]; closed triangles = low dose of NE [200nM]) and salbutamol-treated (closed hexagons) rats presented as a percentage of the Control value. Peak responses generally occurred between 60 and 90 minutes during the hypoglycemic clamp procedure. The bars indicate the mean ± SEM for each treatment group. Both NE and salbutamol treatment significanty reduced peak epinephrine responses compared to Controls (P < 0.05 and P < 0.0001, respectively). Inhibiting lactate transport in NE-treated animals using 4CIN restored peak epinephrine responses to normal (P < 0.05 vs NE).

To establish whether the elevated VMH lactate levels caused by the antecedent NE treatment was responsible for suppressing the counterregulatory responses, we administered 4CIN to NE-treated rats just prior to the start of the hypoglycemic clamp. Blocking neuronal lactate uptake with 4CIN improved, but did not fully restore, the glucagon response. However, 4CIN fully restored the epinephrine responses to normal in NE-treated animals. This response was specific, as the administration of 4CIN alone did not further augment the counterregulatory hormone responses in control animals (Supplemental Figure 3 (43)).

Immunohistochemistry

To better understand where NE was acting within the VMH, we identified the cell populations in the VMH that expressed β2-adrenergic receptors using immunohistochemistry. We stained for β2-adrenergic receptors and either the astrocyte marker, glial fibrillary acid protein (GFAP), or the neuronal marker, NeuN. The β2-adrenergic receptors co-localized with both GFAP and NeuN, indicating β2-adrenergic receptors were expressed on both VMH astrocytes and neurons (Fig. 6). The co-localization observed in neurons was more abundant than in the astrocytes.

Figure 6.

β2-adrenergic receptors are expressed on both astrocytes and neurons in the VMH. Representative immunohistochemical images depicting staining of β2-adrenergic receptors (β2AR; green) and either NeuN, a neuronal marker (in red; top row) or glial fibrillary acid protein (GFAP; in red; bottom row) in the ventromedial hypothalamus (VMH). DAPI counterstain is in blue. The merged image shows co-localization of β2-adrenergic receptors with both NeuN and GFAP in yellow. The panel on the far right is a magnified image of the square outline with the scale bar indicating a length of 50 μm (top row) or 20 μm (lower row). 3V = third ventricle.

Discussion

The adrenergic system is important for the recovery from acute hypoglycemia—centrally, the activation of adrenergic receptors helps to augment the release of counterregulatory hormones (12, 13) whereas systemically, epinephrine enhances hepatic glucose production and limits glucose utilization by peripheral tissues (44, 45). These mechanisms have been well documented. However, the effects of recurring activation of adrenergic receptors, especially those within the brain, are less clear. The current study investigated how repeated activation of VMH noradrenergic receptors affects the counterregulatory hormone responses to hypoglycemia. Our data showed that recurring activation of β2-adrenergic receptors in the VMH led to significant increases in local lactate levels in the interstitial fluid which contributed to blunting of the counterregulatory hormone response to hypoglycemia.

During acute bouts of insulin-induced hypoglycemia, a decrease in ambient glucose levels within the VMH raises local extracellular NE concentrations by ~170% (9-11). Importantly, the rise in VMH NE is dependent on decreases in local extracellular glucose levels, as preventing hypoglycemia in the VMH with a localized glucose perfusion in the face of systemic hypoglycemia, abolishes the rise in VMH NE (11). In addition to the dependence on glucose levels, the interstitial glucose threshold for NE release in the VMH is dependent on the rate at which glucose levels fall. When plasma glucose levels decline rapidly, NE is released at higher glucose concentrations, but when blood glucose levels decline more slowly, VMH NE release occurs at a lower threshold (46-48). While these studies demonstrate that local glucose deprivation activates the VMH noradrenergic system during acute bouts of hypoglycemia, less is known about how recurring exposure to hypoglycemia affects this neurotransmitter system.

De Vries and colleagues reported that prior antecedent hypoglycemia did not significantly impact VMH NE responses during subsequent episodes of hypoglycemia, despite the presence of a markedly suppressed sympathoadrenal response (14, 46). These observations are consistent with the findings from the current study and together they indicate that the site or cause of sympathoadrenal impairment may lie downstream of NE release in the VMH. In healthy human subjects, Ramanathan and colleagues demonstrated that adrenergic blockade prevented the hypoglycemia-induced reduction in catecholamine responses during subsequent bouts of hypoglycemia (15), suggesting repeated activation of the adrenergic system contributes to the development of counterregulatory failure following recurring exposure to hypoglycemia. However, whether the adrenergic effects were mediated centrally or peripherally could not be determined from the earlier study as propranolol acts in both regions. Here, we report for the first time that repeated microinjection of NE directly into the VMH was enough to suppress the counterregulatory hormone responses to hypoglycemia, indicating that repeated activation of these hypothalamic NE receptors contributes to the development of HAAF. Taken together, the data show that while NE release in the VMH remains intact during antecedent episodes of hypoglycemia, repeated activation of adrenergic receptors in the VMH triggers downstream events that dampen the counterregulatory hormone response to hypoglycemia.

To better understand the mechanism(s) by which repeated activation of the VMH NE system mediates its suppressive effects on the counterregulatory response, we identified the subclass of adrenergic receptors that recapitulated the defect. Previous work by Szepietowska and colleagues indicated that β ₂-adrenergic receptors in the VMH were responsible for augmenting the epinephrine response to an acute bout of hypoglycemia (12, 13). We showed that multiple injections of salbutamol, a short-acting β ₂-adrenergic receptor agonist, directly into the VMH recapitulated the counterregulatory defects observed in the NE group. This suggests β2-adrenergic receptors are likely the predominant subclass of adrenergic receptors in the VMH that are responsible for the suppressive effects of recurrent adrenergic activation on counterregulatory hormone release. It is interesting to note that the suppressive effects of salbutamol were more pronounced than with the administration of NE, indicating that NE may act through other noradrenergic receptors in the VMH to modulate the magnitude of counterregulatory hormone responses to hypoglycemia.

To understand where NE was acting within the VMH, we used immunohistochemistry to show that β2-adrenergic receptors are expressed on neurons, as well as astrocytes, within the VMH. It has been reported that stimulation of β ₂-adrenergic receptors can facilitate the spontaneous release of the excitatory neurotransmitter glutamate in VMH neurons using a cAMP/PKA signal transduction pathway, as well as enhance glycogenolysis in cortical astrocytes (21, 25). Importantly, the breakdown of astrocytic glycogen stores results in the production of lactate (20, 21). To assess whether repeated activation of β2-adrenergic receptors in the VMH increases local lactate levels, we measured lactate concentrations in the collected microdialysate samples. Here, we noted that prior treatment with either NE or salbutamol increased extracellular lactate concentrations in the VMH. To determine whether the elevated lactate levels contributed to counterregulatory failure, we inhibited neuronal lactate transport in the VMH using 4CIN in a subgroup of NE-treated animals during the hypoglycemic clamp. Despite the higher lactate levels, inhibiting lactate uptake in neurons improved the glucagon response and restored the sympathoadrenal response to normal indicating NE activates downstream mechanisms that enhance lactate production, which in turn, attenuate the counterregulatory hormone response. The high lactate concentrations noted in NE and salbutamol-treated animals is consistent with this idea. This observation is significant in light of the fact that local delivery of lactate into the VMH suppresses the counterregulatory response to hypoglycemia in nondiabetic, hypoglycemia-naïve rats and that inhibition of lactate transport also restores the counterregulatory response in recurrently hypoglycemic rats where VMH lactate concentrations are elevated (8, 26). Lactate is a critically sensed fuel substrate in the brain that regulates feeding and metabolic processes either through its uptake and metabolism in neurons or as a signaling molecule (49-53). Under the current conditions, whether lactate serves as a fuel substrate or as a signaling molecule in the VMH is less clear, but reversal of the counterregulatory defect through the inhibition of lactate transport favors the former. In support of this notion, we previously reported that the suppressive effects of local lactate delivery on the counterregulatory response to hypoglycemia can also be reversed by pharmacological inhibition of lactate dehydrogenase in the VMH (26).

Furthermore, Wiegers and colleagues observed a greater fall in brain lactate levels in the brains of type 1 diabetic patients with impaired awareness of hypoglycemia compared to both healthy and type 1 diabetic patients with normal awareness of hypoglycemia, suggesting that the brains of type 1 diabetic patients with impaired awareness of hypoglycemia oxidize more lactate during hypoglycemia and this may account for loss of hypoglycemia symptoms. This is consistent with the conclusions drawn in the current study, which shows that inhibition of lactate uptake (and presumably its oxidation) restored the defects in counterregulatory hormone secretion. In contrast to these findings, De Feyter and colleagues reported that brain lactate concentrations were elevated in patients with type 1 diabetes, although no increase in brain lactate oxidation was noted during hypoglycemia (28). The differences between these studies may be attributed to the methodology that was used to ascertain brain lactate levels. Moreover, the scenario in type 1 diabetes is much more complex and likely involves a multitude of factors, including differences in metabolic control between subjects (ie, poor metabolic control likely results in higher brain lactate levels), as well as the frequency, depth, and rate of hypoglycemia development experienced by each individual can all influence the extent of adrenergic activation (11, 46). Importantly, the latter can influence the development of HAAF as noted by Lontchi-Yimagou et al (16)—subjects with larger epinephrine responses during antecedent episodes of hypoglycemia developed HAAF, whereas those with smaller or no epinephrine responses, did not develop HAAF. Together, these studies further underscore the importance of central lactate in relation to the development of counterregulatory failure and hypoglycemia unawareness.

The current study demonstrates there are indeed long-lasting effects of recurring adrenergic activation on lactate production and/or utilization within the VMH, but the precise mechanism(s) linking the 2 phenomena still need to be worked out. Whether the rise in VMH lactate levels following antecedent NE administration is mediated directly through the activation of adrenergic receptors on astrocytes or indirectly through stimulation of other neuronal pathways within the VMH is not clear (20, 25, 54). And while identification of these downstream mechanisms is beyond the scope of the current study, studies are currently underway in our laboratory to investigate these mechanisms in more detail.

Conclusion

We therefore conclude that repeated activation of VMH noradrenergic receptors and more specifically, β2-adrenergic receptors, can lead to changes in downstream mechanisms that enhance lactate production and ultimately, lead to suppression of the counterregulatory hormone response to hypoglycemia. These findings provide new insight into the possible pathophysiological mechanisms that lead to a blunting of the counterregulatory response following recurrent exposure to hypoglycemia. In addition, this study identifies a novel mechanism that helps bring together several observations in this field of study: (1) that the VMH noradrenergic system is activated in response to hypoglycemia; (2) that VMH NE responses are not affected by recurrent exposure to hypoglycemia despite the presence of counterregulatory failure; and (3) repeated activation of the adrenergic system leads to counterregulatory failure. Together, the data have important implications for the treatment or prevention of hypoglycemia in patients with type 1 diabetes, as we recently reported that treatment with low doses of the nonspecific β-adrenergic receptor blocker carvedilol improved the counterregulatory hormone response and hypoglycemia awareness in recurrently hypoglycemic rats (55). These improvements were associated with a reduction in central lactate concentrations.

Abbreviations

Abbreviations
 
• 4CIN

  alpha-4-hydroxycinnamic acid

 
• aECF

  artificial extracellular fluid

 
• GFAP

  glial fibrillary acid protein

 
• HAAF

  hypoglycemia-associated autonomic failure

 
• NE

  norepinephrine

 
• NeuN

  neuronal nuclear protein

 
• PBS

  phosphate-buffered saline

 
• VMH

  ventromedial hypothalamus

Acknowledgments

The authors would like to thank Aida Groszmann, Codruta Todeasa, Maria Batsu, and Ralph Jacob for their invaluable assistance with the hormone assays in these studies and Dr. Michael Bridge from the University of Utah’s Cell Imaging Core for his assistance with the immunohistochemical imaging.

A.S., W.Z., P.W., R.F., N.K., D.A., and O.C. researched the data. A.S. wrote the manuscript. O.C. contributed to protocol development and reviewed/edited the manuscript. All authors have reviewed and approved the manuscript.

Financial Support: This work was generously supported by research grants from the Juvenile Diabetes Research Foundation (3-SRA-2017-487-S-B) and the National Institutes of Health (R01 DK099315).

Additional Information

Disclosures: No potential conflicts of interest relevant to this article were reported.

Dr. Owen Chan is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data Availability

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.