Role of dopamine in the development of impaired counterregulation and impaired awareness of hypoglycemia

Erica Macon; Micah H Devore; Adriana Vieira de Abreu; Rahul Agrawal; Sunny Sharma; Parker Howe; Ashlee Marksbury; Mason Wooten; Evan Brockman; Owen Chan; Simon J Fisher

doi:10.1016/j.xcrm.2026.102710

. 2026 Mar 27;7(4):102710. doi: 10.1016/j.xcrm.2026.102710

Role of dopamine in the development of impaired counterregulation and impaired awareness of hypoglycemia

Erica Macon ^1,^3,⁴, Micah H Devore ^1,⁴, Adriana Vieira de Abreu ², Rahul Agrawal ², Sunny Sharma ², Parker Howe ², Ashlee Marksbury ¹, Mason Wooten ¹, Evan Brockman ¹, Owen Chan ², Simon J Fisher ^1,^5,^∗

PMCID: PMC13130643 PMID: 41903547

Summary

For people with diabetes, the syndrome of hypoglycemia-associated autonomic failure (HAAF) is composed of a blunted counterregulatory response (CRR) and impaired awareness of hypoglycemia (IAH). The objective herein is to identify a drug that might correct these components of HAAF. A rodent model of HAAF was developed to induce both impaired CRR and IAH (blunted food intake response to hypoglycemia). A drug screen identifies the dopamine antagonist, metoclopramide (MET), that prevents the development of HAAF. Additionally, following the induction of HAAF, MET restores normal awareness and CRRs. Conversely, the dopamine receptor agonist (bromocriptine), when administered centrally, induces HAAF. Finally, dopaminergic modulation of the ventral tegmental area (VTA) induces reciprocal changes in CRR to hypoglycemia with matching changes in VTA dopaminergic gene expression. These data identify dopamine signaling as a critical mediator of HAAF and dopamine antagonism as a potential treatment strategy.

Keywords: insulin, brain, cognition, metabolism, diabetes, dopamine, metoclopramide, hypoglycemia, counterregulation, rat

Graphical abstract

Highlights

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A rodent model of hypoglycemia-associated autonomic failure (HAAF) was developed
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A dopamine antagonist restores the hormonal and awareness response to hypoglycemia
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Conversely, a dopamine agonist is sufficient to induce HAAF
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Dopamine action in a specific brain area may mediate these responses to hypoglycemia

Macon and Devore et al. developed a rodent model of hypoglycemia-associated autonomic failure (HAAF). They demonstrate that the dopamine antagonist, metoclopramide, restores normal awareness and counterregulatory responses to hypoglycemia. These findings identify dopamine signaling as an important mediator of HAAF and dopamine antagonism as a potential treatment strategy for HAAF.

Introduction

Hypoglycemia is the most common clinical complication in diabetes management and poses a major obstacle in attempts to optimally lower blood sugar levels.¹ Clinically, it has been well established that recurrent episodes of hypoglycemia lead to the development of hypoglycemia-associated autonomic failure (HAAF), characterized by a blunted sympathoadrenal response to hypoglycemia and impaired awareness of hypoglycemia (IAH).²^,³^,⁴^,⁵^,⁶^,⁷ Although IAH and defective counterregulatory responses (CRRs) do not necessarily develop in parallel, patients with type 1 diabetes mellitus (T1DM) exhibit IAH even in the absence of proven HAAF, thus highlighting the negative impact on quality of life for these patients.⁸ In spite of the implementation of advanced diabetes technologies, IAH remains a serious problem for people with diabetes.⁹^,¹⁰^,¹¹ The burden of IAH is undoubtedly underestimated as people with diabetes who report IAH often demonstrate impaired awareness of biochemically confirmed hypoglycemia.¹² Although as little as one bout of hypoglycemia can blunt the subsequent response to hypoglycemia,⁵^,¹³^,¹⁴ the underlying mechanisms by which HAAF develops remain elusive and may be multifactorial.²^,¹⁵ Ultimately though, impaired brain glucose sensing and impaired central nervous system (CNS) neurotransmitters release in response to hypoglycemia is characterized in HAAF by a failure to fully activate the autonomic nervous system necessary for appropriate counterregulatory and awareness responses.²^,¹⁶^,¹⁷

A major obstacle in the field of IAH research has been the lack of a suitable animal model with which to study the condition. Aside from rodents, previous research has utilized zebrafish, pig, and monkeys¹⁸^,¹⁹^,²⁰ to investigate hypoglycemia, yet the etiology between IAH and diabetes remains unresolved. The major reason for this hindrance is the difficulty in objectively assessing the classic symptoms of hypoglycemia (anxiety, perspiration, hunger, confusion, drowsiness, weakness) in animal models. It could be argued that hunger is the most clinically relevant symptom of hypoglycemia since hunger prompts the initiation of a potentially life-saving food-seeking behavioral response. Fortunately, food intake can be easily measured in rodents. Indeed, acute insulin-induced hypoglycemia has been shown to increase hunger and food intake in humans²¹^,²²^,²³^,²⁴^,²⁵ and rodents.²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³² Similarly, acute glucoprivation induced with 2-deoxyglucose (2DG) in rodents also increases food intake 4-fold.³³ Thus, the increase in food intake associated with hypoglycemia or glucoprivation is a good surrogate marker for hunger and awareness of hypoglycemia. In attempts to establish an animal model of IAH, recurrent hypoglycemia (RH) reproducibly induces a blunted sympathoadrenal response to hypoglycemia, but RH does not induce a blunted food intake response to hypoglycemia.²⁶^,³⁴ Interestingly though, recurrent 2DG (R2DG) administration does induce a blunted food intake response.³³

In terms of identifying the specific neurotransmitter responses that are impaired in HAAF, there is evidence for altered CNS GABAergic (gamma-aminobutyric acid)³⁵^,³⁶ and glutaminergic³⁷ signaling; however, the role for other neurotransmitters in the development of HAAF has not been fully characterized. Previous work in the field has suggested that altered dopaminergic signaling might be involved in glucoregulatory and whole-body homeostasis in animal models of T1DM³⁸^,³⁹ and T2DM.⁴⁰^,⁴¹^,⁴²^,⁴³ Acute alterations in brain glucose levels rapidly modify the release of dopamine.⁴⁴ In response to insulin-induced hypoglycemia, levels of dopamine increase significantly in the ventromedial hypothalamus (VMH)⁴⁵ and striatum.⁴⁶ Consistent with an important role for acute increases in dopamine release in mediating awareness of hypoglycemia, it has been reported that the food intake responses to hypoglycemia are abolished in dopamine-deficient⁴⁷ and dopamine receptor knockout⁴⁸ mice. Following recurrent episodes of iatrogenic hypoglycemia, dopaminergic receptor mRNA is down-regulated as if in response to repetitive dopaminergic signaling.⁴⁹ Clinically, antecedent treatment of healthy men with the dopamine receptor agonist bromocriptine (BROMO) blunted the neuroendocrine response to insulin-induced hypoglycemia.⁵⁰ Taken together, these results indicate an important role for dopamine as a neurotransmitter that participates in the response to acute hypoglycemia. If recurrent dopamine activation leads to the development of HAAF, it would be logical to test whether dopamine receptor blockade prevents the development of HAAF. Therefore, the current study performed detailed experiments using agonists and antagonists of the dopamine receptor to (1) identify therapeutic treatments that improve both the IAH and impaired counterregulatory components of HAAF and (2) determine the site and mechanism by which these agents achieve their beneficial effects.

Results

Development of IAH rodent model

With a goal of establishing a model of IAH, a reproducible measurement of hypoglycemia awareness was first identified. Rats were given either a singular Neutral Protamine Hagedorn (NPH) insulin ([INS], 15 units/kg body weight [BW] subcutaneously [s.c.]) or saline ([SAL], 1 mL/kg s.c.) injection and had their blood glucose (BG) concentrations and food intake monitored for 6 h. By experimental design, BG for the INS-treated rats was significantly lower compared to that for SAL-treated rats (p = 0.0074) (Figure 1A). Food intake was higher for the INS-treated (5.6 ± 0.3 g/6 h) vs. SAL (1.9 ± 0.4 g/6 h) (p < 0.0001) (Figure 1B). For the purposes of this paper, this quantifiable increase in food intake in rodents in response to insulin-induced hypoglycemia will be referred to as awareness of hypoglycemia.

RH has been a well-established method to induce a blunted CRR in rodents,⁵¹^,⁵² but it is unclear whether 3 days of RH preconditioning would similarly induce a blunted food intake response to a subsequent episode of acute insulin-induced hypoglycemia on day #4 (Figure 1C). Two control groups were injected with saline (SAL 1 mL/kg s.c.) daily for three recurrent days (RS). Another group of rodents was injected daily with insulin (NPH, day 1 RH: 2.0 U/kg, day 2 RH: 1.5 U/kg, day 3 RH: 1.2 U/kg s.c.) to induce recurrent episodes of hypoglycemia (RH) (Figure S1). On day 4, one recurrent saline group received another saline injection (RS + SAL; 1 mL/kg s.c.), whereas the other recurrent saline-treated group received an INS injection (RS + INS; NPH 15 U/kg s.c.), and the RH-preconditioned rodents received a fourth day of insulin (RH + INS; NPH 15 U/kg s.c.) (Figure 1C). As compared to saline-injected controls (RS + SAL), a single injection of insulin on day 4 resulted in equivalent hypoglycemia and an equal increase in food intake in animals preconditioned with either recurrent saline (RS + INS) or RH (RH + INS) (Figures 1D and 1E). Food intake in RH + INS (5.13 ± 0.2 g) was not significantly different than RS + INS (3.9 ± 0.4 g), yet both were 5- to 6-fold higher than euglycemic, saline-treated RS + SAL (0.78 ± 0.3 g) (p < 0.0001) (Figure 1E). Thus, RH preconditioning did not blunt the rats’ awareness of hypoglycemia.

Since R2DG (200 mg/kg/day s.c.) induces a blunted food intake response to subsequent 2DG administration,³³ we tested whether R2DG would also blunt the food intake response to subsequent insulin-induced hypoglycemia. R2DG was administered daily for 3 day and compared to two recurrent saline-injected control groups (RS; 1 mL/kg/day s.c.). On day 4, along with the R2DG group, one recurrent saline group received an INS injection to induce hypoglycemia (RS + INS; NPH 15 U/kg s.c.), while the other received another SAL injection (RS + SAL) (Figure 1F). By experimental design, the two insulin-treated groups had lower blood sugars (p < 0.0001) than the saline-injected group (RS + SAL) (Figure 1G). As expected, insulin-induced hypoglycemia resulted in a marked increase in food intake (RS + INS; 3.9 ± 0.6 g/4 h) above saline controls (SAL+RS; 1.2 ± 1.1 g/4 h). In contrast, antecedent R2DG treatment resulted in a blunted food intake response to hypoglycemia to levels not different than saline controls (R2DG + INS; 2.1 ± 0.3 g/4 h; p = 0.92 vs. RS + SAL) (Figure 1H).

When experiments were pooled for analysis, food intake was strongly correlated with decreasing BG levels in RS + INS as compared to RS + SAL (Figure 1I, slope “m” = −0.0066, r = 0.58, p < 0.0001). This strong correlation between food intake and decreasing BG was maintained after RH preconditioning (Figure 1J, m = −0.0078, r = 0.91, p < 0.0001) indicating RH preconditioning did not blunt the rats’ awareness of hypoglycemia. In contrast, the increased food intake response to hypoglycemia was markedly blunted following R2DG preconditioning (Figure 1K, m = −0.0011, r = 0.16, p = NS). This blunted food intake response to insulin-induced hypoglycemia reflects an impaired awareness to hypoglycemia.

It has been well established that the neuroglycopenia induced with acute 2DG (200 mg/kg s.c.) treatment elicits a CRR and a spike in blood sugar levels (Figure 1L). Of note, this increased blood sugar response to 2DG was significantly blunted with sequential days of 2DG treatment (day 1 glucose post-2DG: 253 ± 6 mg/dL; day 2 glucose: 176 ± 10 mg/dL; day 3 glucose: 148 ± 6 mg/dL) compared to saline controls (97 ± 3 mg/dL) (p < 0.02; Figure 1L).

Since profound hypoglycemia can cause brain damage,⁵³^,⁵⁴^,⁵⁵ experiments were conducted to determine if 2DG treatment caused brain damage. Rats were either given 2DG (R2DG; 200 mg/kg/day s.c.) or saline (SAL; 1 mL/kg s.c.), and 7 days later, the brain tissue was sectioned and stained for Fluoro-Jade B, a marker for neuronal degeneration. The number of neurons positively stained with Fluoro-Jade B were lower in R2DG (182 ± 30 positive neurons) compared to SAL (343 ± 26 positive neurons; p = 0.0014) (Figure 1M). Furthermore, to determine if the 2DG effect was reversible or resulted in a long-term blunting of the food intake response to hypoglycemia, three experimental groups were investigated: (1) RS + INS (saline 1 mg/kg, NPH 15 U/kg s.c.), (2) R2DG + INS (2DG 200 mg/kg/day, NPH 15 U/kg s.c.), and (3) R2DG + 2-week recovery + INS (2DG 200 mg/kg/day, NPH 15 U/kg s.c.). By experimental design, all groups received an INS injection, which resulted in hypoglycemic levels that were not different from each other (p = 0.78; Figure 1N). As expected, following 3 days of R2DG the food intake response to insulin-induced hypoglycemia was reduced in R2DG + INS (3.0 ± 0.5 g/4 h) compared to RS + INS (5.1 ± 0.3 g/4 h, p < 0.05) (Figure 1O). Two weeks following the three-day R2DG conditioning period, the food intake response (6.1 ± 0.7 g/4 h) to insulin-induced hypoglycemia was not different from RS + INS.

Metoclopramide increases food intake during hypoglycemia

Food and Drug Administration-approved drugs were screened to determine if they could increase food intake response to hypoglycemia. From this screen, the dopamine antagonist metoclopramide (MET) was noted to increase food intake during hypoglycemic conditions. As noted in Figure 2A, rodents treated with an acute injection of SAL (1 mL/kg s.c.) remained euglycemic (98 ± 2 mg/dL) for 6 hours. In rodents treated with insulin alone (INS; NPH 15 U/kg s.c. BW) or with both INS and MET (NPH 15 U/kg s.c., MET 3 mg/kg intraperitoneally [i.p.], respectively), BG concentrations dropped to a similar extent (INS 37 ± 5 mg/dL; INS + MET 40 ± 5 mg/dL) (p < 0.0001 vs. SAL treatment; Figure 2A). Food intake was higher for insulin-induced hypoglycemic groups compared to the SAL (1.4 ± 0.3 g/6 h) group (p < 0.01). Additionally, the MET + INS (5.7 ± 0.3 g/6 h)-treated group had higher food intake responses compared to the INS (4.3 ± 0.2 g/6 h) group (p < 0.05; Figure 2B).

MET prevents the development of hypoglycemia unawareness and restores awareness of hypoglycemia

(A–D) MET effects on hypoglycemia induced food intake. (A) BG levels following an acute injection of saline (SAL, n = 6) or insulin (INS, n = 6; INS + MET, n = 8) (15 U/kg BW), (∗p < 0.0001 SAL vs. INS, ∗p < 0.0001 INS+MET vs. SAL). Groups were compared via two-way ANOVA. (B) Cumulative food intake after an acute injection of saline, insulin (15 U/kg BW), or insulin + MET (∗∗p < 0.01 INS vs. SAL; ∗∗∗∗p < 0.0001 INS + MET vs. SAL; ∗p < 0.05 INS + MET vs. INS). Groups were compared via one-way ANOVA. (C) BG levels following a singular injection of saline (SAL, n = 3) or MET (n = 4). Groups were compared via two-way ANOVA. (D) Cumulative food intake under euglycemic conditions for MET and SAL. Groups were compared via unpaired t test. Each bar represents the mean + SEM.

(E–G) MET prevented the development of IAH. (E) Schematic diagram of experimental design using the prevention protocol of IAH with R2DG (RS + SAL, n = 6; RS + INS, n = 6; R2DG + INS, n = 6, R2DG + MET + INS, n = 8). (F) BG levels following 3 days of RS, R2DG, or R2DG + MET (3 mg/kg/d) followed by one single injection of saline or INS (NPH 15 U/kg) on day 5 (∗p < 0.0001 RS + SAL vs. RS + INS, R2DG + INS, and R2DG + MET + INS). Groups were compared via two-way ANOVA. (G) Cumulative food intake (following preconditioning period) in response to a single saline or INS injection (∗∗∗∗p < 0.0001 RS + SAL vs. RS + INS; ∗∗∗∗p < 0.0001 RS + INS vs. R2DG + INS; ∗∗p < 0.01 R2DG + INS vs. R2DG + MET + INS). Groups were compared via two-way ANOVA. Each bar represents the mean + SEM.

(H–J) MET restored hypoglycemia awareness. (H) Schematic diagram of experimental design of treatment protocol of IAH (RS + SAL, n = 8; RS + INS, n = 6; R2DG + INS, n = 6, R2DG + MET + INS, n = 6). (I) BG levels following 6 days of RS, R2DG, or R2DG + MET (3 mg/kg/d) injections and one single injection of saline or INS (NPH 15 U/kg) on day 7 (∗p < 0.0001 between groups). Groups were compared via two-way ANOVA. (J) Cumulative food intake (following preconditioning period) in response to a single saline or INS injection (∗∗∗∗p < 0.0001 RS + SAL vs. RS + INS & R2DG+MET+INS; ∗∗∗p < 0.001 RS + INS vs. R2DG + INS; ∗∗p = 0.002 R2DG + INS vs. R2DG+MET+ INS). Groups were compared via one-way ANOVA. Each bar represents the mean + SEM.

(K–M) MET prevents IAH in diabetic model. (K) Schematic diagram of experimental design of treatment protocol of IAH in diabetic rats (STZ + RS + INS, n = 6; STZ + R2DG + INS, n = 4; STZ + R2DG + MET + INS, n = 6). Prior to the prevention protocol using MET, rats underwent vascular surgery, recovery, and STZ injection. (L) Blood glucose levels in diabetic rats during a hyperinsulinemic-hypoglycemic clamp. (M) Cumulative food intake (following preconditioning period) during the hypoglycemic clamp (#p = 0.02 STZ + RS + INS vs. STZ + R2DG + INS; ∗p = 0.01 STZ + R2DG + INS vs. STZ + R2DG + MET + INS). Groups were compared via one-way ANOVA. Each bar represents the mean + SEM.

To determine if the observed MET augmentation of the food intake response was unique to conditions of hypoglycemia, food intake responses were quantified under euglycemic conditions. Under euglycemic conditions (Figure 2C), food intake in response to SAL (saline 1 mL/kg s.c., 1.7 ± 0.5 g/4 h) was not different than in response to MET (3 mg/kg i.p., 1.2 ± 0.2 g/4 h) (p = 0.54; Figure 2D).

Metoclopramide both prevents the development of IAH and restores awareness of hypoglycemia in an IAH model

Two protocols with MET administration were examined: first, whether MET treatment could prevent the development of IAH and second, whether MET treatment could restore awareness in rodents that had established IAH.

In the MET prevention protocol, MET (3 mg/kg/day i.p.) was administered daily, prior to the administration of 2DG (200 mg/kg/day s.c.) and compared to SAL (1 mL/kg s.c.)-injected animals (Figure 2E). By experimental design, all groups that received insulin (NPH 15 U/kg s.c.) on the final day had lower BG compared to the saline-treated (RS + SAL) group (p < 0.0001; Figure 2F). As expected, insulin-induced hypoglycemia (RS + INS) resulted in higher food intake (5.7 + 0.2 g/4 h) than saline-injected controls (RS + SAL; 1.9 + 0.2 g/4 h, p < 0.0001) (Figure 2G). In spite of achieving similar levels of hypoglycemia, recurrent treatment with 2DG (R2DG + INS) resulted in a blunted food intake response (1.9 ± 0.6 g/4) to insulin-induced hypoglycemia, which was significantly lower than food intake in the RS + INS group (p < 0.0001). However, in the MET-treated group (R2DG+MET+INS), the food intake response (4.2 ± 0.4 g/4 h) was higher compared to the R2DG + INS group (p < 0.01).

In the MET restoration protocol (Figure 2H), two groups of animals received daily injections of 2DG (200 mg/kg/day s.c.) to induce the blunted food intake response noted in the previous three-day R2DG protocols; however, on days 4–6, these rodents were randomized to continued R2DG treatment or randomized to MET (3 mg/kg/day i.p.) administration immediately before continued daily R2DG treatment. These two treatment groups were compared to saline (1 mL/kg s.c.)-injected control rodents (Figure 2H). By experimental design, all groups that received insulin (NPH 15 U/kg s.c.) on the final day had lower BG (RS + INS 40 ± 5 mg/dL; R2DG + INS 48 ± 11 mg/dL; R2DG + MET + INS 55 ± 10 mg/dL) compared to the RS + SAL (124 ± 3 mg/dL)-treated group (Figure 2I). Food intake responses were significantly different between groups in the restoration model (p < 0.0001) (Figure 2J). As compared to saline-treated control (RS + SAL 1.8 ± 0.2 g/4 h), food intake was increased with insulin-induced hypoglycemia (SAL+INS 4.9 ± 0.4 g/4 h) and was lower with antecedent recurrent 2DG treatment (R2DG + INS 2.7 ± 0.4 g/4 h) (p < 0.001 vs. SAL+INS), indicating that R2DG blunted the food intake response to hypoglycemia. Following 3 days of recurrent 2DG treatment, subsequent MET treatment resulted in a higher food intake response (R2DG + MET + INS 4.8 ± 0.2 g/4 h) compared to R2DG + INS (p = 0.002). MET treatment fully restored the food intake response to hypoglycemia to a level not different from RS + INS-treated group (p = 0.99).

Noting that antecedent treatment with MET prevented the development of IAH in non-diabetic rodents, this beneficial effect of MET was re-examined in a streptozotocin ([STZ], 65 mg/kg i.p.) insulin-deficient rodent model of type 1 diabetes (Figure 2K). STZ-diabetic rats were treated with recurrent daily saline injections (RS 1 mL/kg s.c.) or with R2DG (200 mg/kg/day i.p.) injections (with or without MET [3 mg/kg/day i.p.). Following the antecedent induction protocols, rats underwent hyperinsulinemic-hypoglycemic clamp. The net food intake was assessed over the 5-hour clamp period. All three treatment groups were maintained in the hypoglycemic range for ∼285 min (Figure 2L). During the last 90 min of the clamp, BG values were not different for STZ + R2DG + INS and STZ + R2DG + MET + INS (48 ± 1.6 and 46 ± 4.8 mg/dL, respectively, p = NS). However, BG for STZ + RS + INS (67 ± 2.7 mg/dL) was significantly different than that for the two other groups (p < 0.01). Food intake responses were different between treatment groups (p = 0.02) with STZ + R2DG + INS (4.0 ± 0.5 g/5 h) having the lowest response (i.e., blunted response) compared to the control STZ + RS + INS (6.5 ± 0.6 g/5 h). Antecedent MET treatment (STZ + R2DG + MET; 6.6 ± 0.4 g/5 h) restored the food intake response to hypoglycemia to levels not different from the control (STZ + RS + INS) group (Figure 2M).

Prevention of a blunted CRR and restoration of the CRR with MET treatment

Using this model of IAH (achieved with R2DG [200 mg/kg/day s.c.] preconditioning), the CRR to hypoglycemia with and without MET (3 mg/kg/day i.p.) was examined in non-diabetic rodents (Figure 3A). On the final day of the prevention protocol, all treatment groups (RS + INS, R2DG + INS, and R2DG + MET + INS) underwent a hyperinsulinemic-euglycemic-hypoglycemic clamp where BG concentrations were maintained initially at euglycemic levels and then hypoglycemic levels for 90 min. BG concentrations during the clamp did not differ between groups (p = 0.14; Figure 3B). Epinephrine and glucagon concentrations were quantified at basal and did not differ between any group (p < 0.07). Peak epinephrine levels during hypoglycemia rose markedly in response to insulin RS + INS (1,642 ± 439 pg/mL), and this epinephrine response was nearly completely abrogated by antecedent treatment with R2DG (R2DG + INS; 211 ± 77 pg/mL). In the setting of R2DG treatment, antecedent MET treatment resulted in a restoration of the epinephrine response (1,373 ± 644 pg/mL, p = 0.04 vs. R2DG + INS) to levels not different than RS + INS (Figure 3C). Glucagon levels showed a similar pattern of responses, although not statistically different between treatment groups (p > 0.09; Figure 3D).

MET restores counterregulation and prevents a blunted counterregulatory response

(A–D) MET prevents blunted CRR in the R2DG model. (A) Schematic diagram of experimental design of prevention of a blunted CRR response using R2DG protocol and treatment with MET. (B) BG concentrations during hyperinsulinemic-euglycemic-hypoglycemic clamp where an infusion of INS (50 mU·kg−1·min−1) and the variable dextrose (40% [BW/vol]) was started (i.e., at −150 min) to induce euglycemia (90–110 mg/dL) and after 120 min of euglycemia, glucose infusion was decreased to induce hypoglycemia (40–50 mg/dL for 90 min). RS + INS, n = 13; R2DG + INS, n = 7; R2DG + MET + INS, n = 9. Groups were measured via two-way ANOVA. (C) Peak epinephrine concentrations at basal and hypoglycemia for all experimental groups (∗p = 0.005 RS + INS (n = 8) vs. R2DG + INS [n = 7]; #p = 0.04 R2DG + INS vs. RS + INS and R2DG + MET + INS [n = 6]). Groups were compared via two-way ANOVA. (D) Peak glucagon concentrations at basal and hypoglycemia for all experimental groups (p > 0.09). RS + INS, n = 9; R2DG + INS, n = 5; R2DG + MET + INS, n = 7. Groups were compared via two-way ANOVA. Each bar represents the mean + SEM.

(E–J) MET prevents blunted CRR in the RH model. (E) Schematic diagram of experimental design of prevention of a blunted CRR response using RH protocol and treatment with MET. RS + INS, n = 12; RH + INS, n = 15; RH + MET + INS, n = 16. (F) BG concentrations during hyperinsulinemic-hypoglycemic clamp where BG is held at 40–45 mg/dL for 90 min. Groups were compared via two-way ANOVA. (G) Glucose infusion (Ginf.) rate during clamp (∗p < 0.001 RS + INS vs. RH + INS and RH + MET + INS; #p = 0.002 RH + INS vs. RH + MET + INS). Groups were compared via two-way ANOVA. (H) Peak epinephrine concentrations at basal and hypoglycemia (∗p < 0.05 RH compared to SAL and RH + MET). Groups were compared via one-way ANOVA. (I) Peak norepinephrine concentrations at basal and hypoglycemia for all experimental groups. No significant difference between groups. Groups were compared via two-way ANOVA. (J) Peak glucagon concentrations at basal and hypoglycemia (∗p < 0.05 RH vs. RS and #p < 0.05 RH vs. RH + MET). Groups were compared via one-way ANOVA. Each bar represents the mean + SEM.

(K–P) MET restores CRR in the RH model. (K) Schematic diagram of experimental design of restoration of the CRR response using the RH protocol and with MET treatment; RS + INS, n = 7; RH + INS, n = 7; RH + MET + INS, n = 7. (L) BG concentrations during hyperinsulinemic-hypoglycemic clamp where BG is held at 40–45 mg/dL for 90 min. Groups were compared via two-way ANOVA. (M) Ginf. rate during clamp (∗p = 0.02 RH + MET + INS vs. RH + INS). Groups were compared via two-way ANOVA. (N) Peak epinephrine concentrations at basal and hypoglycemia (∗p < 0.05 RH compared to SAL and RH + MET). Groups were compared via one-way ANOVA. (O) Peak norepinephrine concentrations at basal and hypoglycemia for all experimental groups. No significant difference between groups. Groups were compares via two-way ANOVA. (P) Peak glucagon concentrations at basal and hypoglycemia. Groups were compared via one-way ANOVA. Each bar represents the mean + SEM.

To determine if MET treatment could also prevent the development of a blunted CRR in another model of HAAF, treatment with or without MET (3 mg/kg/day i.p.) was examined in the 3 days of RH (regular insulin, day 2: 2 U/kg, day 3: 1.5 U/kg, day 4: 1.2 U/kg s.c.) model (Figure 3E, see Figures S2A–S2C for BG conditioning data). After preconditioning with 3 days of recurrent treatment with saline (1 mL/kg s.c.) or insulin-induced hypoglycemia, all groups (RS + INS, RH + INS, and RH+MET+INS) underwent a hyperinsulinemic-hypoglycemic clamp where BG concentrations were fixed in the hypoglycemic range for 90 min. BG concentrations during the clamp were not different between RS + INS (43 ± 0.7 mg/dL) vs. RH + INS (42 ± 0.9 mg/dL) and RH + MET + INS (44 ± 0.5 mg/dL) (p = 0.68; Figure 3F). Glucose infusion rates during hypoglycemia (60–90 min) were higher with RH + INS (32.6 ± 0.5 mg/kg/min) as compared to RS + INS (19.5 ± 0.2 mg/kg/min) and RH + MET + INS (26.3 ± 0.3 mg/kg/min; p < 0.0004) (Figure 3G). MET treatment (RH + MET + INS) resulted in lower glucose infusion rates as compared to RH + INS (p = 0.0002). As expected, insulin-induced hypoglycemia resulted in a marked rise in epinephrine RS + INS (1,499 ± 263 pg/mL) and, as expected in this RH model, the epinephrine response to hypoglycemia was significantly blunted by antecedent RH (RH + INS; 517 ± 117 pg/mL) (p = 0.002) (Figure 3H). Antecedent MET treatment significantly increased epinephrine response (RH + MET + INS 1,076 ± 133 pg/mL) compared to RH + INS (p = 0.02; Figure 3H). Norepinephrine did not increase in response to insulin-induced hypoglycemia (Figure 3I). Peak glucagon concentrations during hypoglycemia rose in response to INS (RS + INS; 64.5 ± 8.5 pg/mL) and was significantly blunted by RH preconditioning (32.6 ± 4.9 pg/mL; p = 0.02). MET prevented the blunting of glucagon secretion (RH + MET + INS; 58.8 ± 7.8 pg/mL) (p < 0.0004 vs. RH + INS; Figure 3J).

It was next determined if MET (3 mg/kg/day i.p.) treatment would restore the CRR in rats with established counterregulatory failure. In this restoration protocol, two groups of animals underwent RH to induce the blunted CRRs noted in the previous 3-day RH protocols; however, on days 4–6 (regular insulin, 1.2 U/kg/day s.c.), these rodents were randomized to continued RH treatment or randomized to MET administration immediately before continued daily RH treatment (see Figures S2D–S2I for BG conditioning data). These two treatment groups were compared to saline (1 mL/kg s.c.)-injected control rodents (Figure 3K). Following this pre-conditioning protocol, RS + INS, RH + INS, RH + MET + INS rats underwent a hyperinsulinemic-hypoglycemic clamp on the final day (Figure 3K). During the hypoglycemic clamp, there were no differences between glucose levels in any groups (p > 0.82; Figure 3L). Glucose infusion rates during hypoglycemia in RH + MET + INS (19.9 ± 1.4 mg/kg/min) were lower compared to RH + INS (26.2 ± 1.4 mg/kg/min) (p = 0.02; Figure 3M). Peak epinephrine concentrations during hypoglycemia were different among treatment groups (p < 0.0001). Insulin-induced hypoglycemia resulted in a marked increase in epinephrine (RS + INS; 1,949 ± 413 pg/mL) that was blunted by antecedent RH (RH + INS; 581 ± 199 pg/mL; p < 0.001 vs. RS + INS) (Figure 3N). Antecedent MET treatment significantly increased the epinephrine response to hypoglycemia (RH + MET + INS; 1,460 ± 202 pg/mL; p = 0.01 vs. RH + INS) (Figure 3N). Norepinephrine did not increase in response to insulin-induced hypoglycemia (Figure 4O). No differences in peak glucagon concentrations during hypoglycemia were detected between any group (p > 0.14; Figure 3P).

Sympathoadrenal response and dopaminergic action with agonist bromocriptine

(A–C) Recurrent BROMO administration in i.c.v. reduced food intake and BG response. (A) Schematic diagram of recurrent aCSF (n = 7) or recurrent BROMO (n = 7, 2 μg) for 3 days into i.c.v. (B) BG on day 4 following singular injection of INS (15 U/kg NPH) Groups were compared via two-way ANOVA. (C) Cumulative food intake after INS injection (∗p < 0.005 RBROMO i.c.v. vs. RCSF i.c.v. + INS). Groups were compared via unpaired t test. Each bar represents the mean + SEM.

(D–H) Recurrent BROMO administration in i.c.v. and effects on CRR. (D) Schematic diagram of recurrent aCSF (n = 7) or recurrent BROMO (n = 7, 2 μg) into i.c.v.. (E) BG during hyperinsulinemic-hypoglycemic clamp where BG is held at 40–45 mg/dL for 90 min. Groups were compared via two-way ANOVA. (F) Ginf. Rate during clamp (∗∗∗∗p < 0.0001 vs. RBROMO). Groups were compared via unpaired t test. (G) Peak epinephrine concentrations at basal and hypoglycemia (∗p < 0.005 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. (H) Peak glucagon concentrations at basal and hypoglycemia (∗p < 0.005 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. Each bar represents the mean + SEM.

(I) Dopaminergic gene expression in VTA. Relative gene product expression of COMT, DDC, DRD2, SLC6A3 in the ventral tegmental area following 3-day treatment with RS (n = 6), RH (n = 7), or RH + MET (n = 6) (∗p < 0.04 RS vs. RH + MET; #p < 0.04 RH vs. RH + MET). Groups were compared via one-way ANOVA. Each bar represents the mean + SEM.

(J–O) Recurrent BROMO infusion in VTA effects on CRR. (J) Schematic diagram of recurrent aCSF (n = 7) or recurrent BROMO (n = 7–9, 5 μg/day; infusion rate 0.1 μL/min). (K) BG concentration during hyperinsulinemic-hypoglycemic clamp, which held BG at 40–50 mg/dL for 90 min. Groups were compared via two-way ANOVA. (L) Ginf. rate during clamp (∗∗∗∗p < 0.001 RCSF vs. RBROMO). Groups were compared via two-way ANOVA. (M) Peak epinephrine concentrations at basal and hypoglycemia (∗p < 0.05 BROMO vs. SAL). Groups were analyzed via unpaired t test. (N) Relative gene expression of DRD2 in the ventral tegmental area following a 3-day treatment with either aCSF or BROMO directly into the VTA (∗p < 0.05 aCSF vs. BROMO). Groups were compared via unpaired t test. (O) Relative gene expression of SLC6A3 in the ventral tegmental area following a 3-day treatment with either aCSF or BROMO directly into the VTA (p = 0.21). Groups were compared via unpaired t test. Each bar represents the mean + SEM.

Sympathoadrenal responses to hypoglycemia with and without the dopaminergic agonist, bromocriptine, and the dopaminergic antagonist, metoclopramide

To provide corroborating evidence that the effects of MET were likely due to the drug’s antagonism of dopamine receptors, experiments were designed to determine if the dopamine agonist, BROMO, would have opposite effects on CRR and hypoglycemia awareness. To assess awareness of hypoglycemia, rats were treated intracerebroventricularly (i.c.v.) for 3 consecutive days with either recurrent daily artificial cerebrospinal fluid (RCSF i.c.v., 1 mL/kg/day) or recurrent daily bromocriptine (RBROMO i.c.v., 2 μg/day) (Figure 4A). On day 4, both groups of rats had a singular insulin injection (NPH 15 U/kg s.c.) and BG, and food intake responses were recorded for 6-h (Figure 4A). BG concentrations during hypoglycemia were not different between treatment groups (p = 0.10; Figure 4B); however, hypoglycemia-induced food intake response was significantly blunted by antecedent recurrent BROMO (RBROMO i.c.v. + INS; 2.5 ± 0.4 g/6 h) treatment compared to antecedent recurrent artificial CSF-treated rats (RCSF+INS; 4.6 ± 0.3 g/6 h) (p = 0.002; Figure 4C).

To determine if recurrent BROMO treatment also blunted the CRR to hypoglycemia, after 3 days of either recurrent daily treatment with RCSF i.c.v. (1 mL/kg/day) or RBROMO i.c.v. (2 μg/day), rats underwent a hyperinsulinemic-hypoglycemic clamp where BG was held at hypoglycemic levels for 90 min (Figure 4D). BG concentrations during the clamp did not differ between groups (p = 0.31; Figure 4E). Glucose infusion rates, however, were higher for RBROMO i.c.v. + INS-treated rats compared to RCSF i.c.v. + INS for the last hour of the clamp (p < 0.0001; Figure 4F). Peak epinephrine (p = 0.03) and glucagon (p = 0.04) concentrations in response to hypoglycemia were blunted in the RBROMO i.c.v. + INS group (439 ± 83 pg/mL and 73 ± 9 pg/mL, respectively) compared to the RCSF i.c.v. + INS-treated group (3,204 ± 1,093 pg/mL and 209 ± 45 pg/mL, respectively) (Figures 4G and 4H).

Based on the results from these series of dopamine antagonist (MET) and dopamine agonist (BROMO) experiments, a role for changes in dopamine gene expression contributing to the impaired CRR to hypoglycemia was examined. Following sacrifice after 3 days preconditioning with saline (RS), RH (regular insulin, day 1: 2 U/kg, day 2: 1.5 U/kg, and day 3: 1.2 U/kg s.c.), or RH and MET (RH + MET, MET, 3 mg/kg/day i.p.), gene expression was examined for dopamine synthesis (DDC), degradation of catecholamines (COMT), regulation of dopamine receptor D2 (DRD2), and synthesis of dopamine transporter (SLC6A3 or DAT). The VMH, substantia nigra, and nucleus accumbens did not show any gene expression differences in any experimental group (p > 0.07; Figures S3A–S3C). In the ventral tegmental area (VTA); however, DRD2 gene expression was lower in the RH-treated group compared to RS, while MET reversed this RH-induced change in gene expression (p < 0.03; Figure 4I). Conversely, RH induced a significant upregulation of SLC6A3, and this effect was reversed with antecedent MET treatment. Given that coordinated changes in dopamine gene expression in the VTA were induced by RH and reversed by MET, the effect of the dopamine agonist BROMO was examined by direct administration into the VTA and the CRR was examined. After 3 days of recurrent BROMO (RBROMO VTA + INS, 5 μg/day) or aCSF (RCSF VTA + INS, 1 mL/kg/day) treatment into the VTA, rats underwent hyperinsulinemic-hypoglycemic clamps (Figure 4J). By experimental design, BG was not different between the groups (p = 0.07; Figure 4K). Glucose infusion rates during the last 40 min of the clamp were higher for RBROMO VTA + INS-treated rats compared to RCSF VTA + INS-treated rats (p < 0.001; Figure 4L). Peak epinephrine concentrations during hypoglycemia were lower for RBROMO VTA + INS (914 ± 71 pg/mL)-treated rats compared to recurrent RCSF VTA + INS (2,006 ± 203 pg/mL)-treated rats (p < 0.05). Additionally, consistent with the RH results, recurrent treatment with BROMO resulted in a decreased gene expression of DRD2 in the VTA (p < 0.05). SLC6A3 gene expression remained unchanged between RCSF and RBROMO (Figure 4O, p = ns). There was no difference in DDC and COMT gene expression in the VTA (Figures S4A and S4B).

Discussion

The current study demonstrates that the increased food intake response to acute insulin-induced hypoglycemia can serve as a surrogate marker for awareness of hypoglycemia. This awareness of hypoglycemia can be blunted by recurrent antecedent treatment with 2DG. Utilizing this rodent model of IAH, a drug screen identified the dopamine antagonist MET as a possible therapeutic agent to improve awareness of hypoglycemia as a component of HAAF. Treatment with MET both before and after the induction of IAH with recurrent 2DG administration, normalized the food intake response to hypoglycemia suggesting restoration of hypoglycemia awareness. Additionally, in both RH and recurrent 2DG models, MET treatment restored the counterregulatory hormonal response to hypoglycemia noted by the near-normal epinephrine and glucagon responses as well as the improved glucoregulatory response (noted by the lower glucose infusion rates observed with MET treatment). Based on this evidence that dopamine antagonism prevented these two principle defects characteristic of HAAF, we inferred that increased dopamine action in the CNS contributed to the development of HAAF. We further examined dopamine’s role in the development of HAAF by demonstrating that treatment with the dopamine receptor agonist, BROMO, was sufficient to induce HAAF as noted by both blunting of hypoglycemia awareness (food intake response to hypoglycemia) and blunting of the CRR to hypoglycemia (noted by blunted epinephrine and glucagon response and higher glucose infusion rates). Finally, these experiments demonstrate that the site of dopamine action during hypoglycemia may be the VTA, as pharmacological agonism and antagonism in this specific area induced reciprocal changes in CRR to hypoglycemia and coordinated changes in dopaminergic gene expression. Collectively, these studies demonstrate that recurrent dopamine agonism leads to the development of HAAF and that dopamine antagonism can prevent the development of HAAF.

Progress in the field of HAAF research has been limited due to the failure to establish a model of IAH. Although other measures of cognitive performance during hypoglycemia have been investigated,⁵⁶^,⁵⁷ awareness of hypoglycemia has not been established in a rodent model. While antecedent RH in rodents consistently induces the blunted CRR to hypoglycemia, we (Figure 1E) and others³⁴ have demonstrated that 3 days of recurrent insulin-induced hypoglycemia does not blunt the food intake response to hypoglycemia (Figures 1E and 1J). It remains unclear why antecedent hypoglycemia reproducibly induces both aspects of HAAF in humans (both a blunted sympathoadrenal response and a blunted symptom response to hypoglycemia)²^,³^,⁴^,⁵^,⁶^,⁷; yet, in rodents, antecedent hypoglycemia only induces a blunted sympathoadrenal response to hypoglycemia. These differential responses may be species specific or may be due to the difficulty in establishing a sensitive test that quantifies awareness of hypoglycemia in rodents.

Consistent with previous studies,³³ sequential days of 2DG treatment induced a blunted glucoregulatory response (Figure 1L). Following recurrent 2DG conditioning, a reproducible blunted food intake response to subsequent insulin-induced hypoglycemia established a model of IAH (Figures 1H, 1K, 1O, 2G, and 2J). It remains unclear why IAH could be induced in rodents with recurrent 2DG administration, but not with recurrent insulin-induced hypoglycemia. We speculated that the neuroglycopenia induced with recurrent 2DG might induce a more profound stimulus to the CNS than RH. However, when investigating more frequent RH preconditioning protocols (6-day protocols and 6-week protocols), these more profound RH protocols also failed to blunt the food intake response to hypoglycemia (data not shown). Thus, while 2DG and hypoglycemia both elicit CRRs, the adaptive (conditioning) responses to recurrent 2DG vs. RH appear to be somewhat different. Of note, the response to 2DG was not related to neuronal damage (Figure 1M). Supporting the evidence that 2DG administration did not cause a permanent neuronal deficit, the adaptive response to recurrent 2DG administration was noted to be reversible (Figure 1O). Regardless of the mechanism for this conditioning response, recurrent 2DG administration consistently induced IAH in the non-diabetic rodents (Figures 1H, 1K, 1O, 2G, and 2J). In the diabetic rodents, even though STZ + RS + INS had slightly higher glucose levels (and presumedly less hypoglycemia stimulus) (Figure 2L), the food intake response remained prominent (Figure 2M). Furthermore, the slightly lower glucose levels in STZ + R2DG + INS (i.e., greater hypoglycemia stimulus) did not augment the food intake response, thus emphasizing the IAH due to recurrent 2DG conditioning. Using this model of IAH,⁵⁸ studies by Farhat et al. also demonstrated that recurrent antecedent 2DG administration suppressed the food intake response to subsequent hypoglycemia.⁵⁹ In the current study, this rodent model of impaired awareness was used to screen for existing compounds that might serve to restore awareness of hypoglycemia (i.e., restore the food intake response to hypoglycemia).

In both the prevention of IAH protocol (Figures 2E and 2K) and treatment of established IAH protocol (Figure 2H), MET administration normalized the food intake response to hypoglycemia, indicating a normalization of hypoglycemia awareness (Figures 2G, 2J, and 2M). While dopaminergic signaling has been reported to modulate food intake and food reward pathways,⁶⁰^,⁶¹^,⁶² it is unlikely that the beneficial effects of MET observed in this study were solely mediated by acute action of MET on food intake. First, in the absence of hypoglycemia, MET treatment did not acutely increase food intake under euglycemic conditions (Figure 2D). Second, MET was only administered during the pre-conditioning period and not on the day of the food intake experiment (Figures 2E and 2H); thus, given MET’s short half-life, it seems unlikely that the noted effects of MET on food intake were mediated a full day after the MET treatment. Finally, the experiments demonstrating the beneficial effects MET in restoring the CRR to hypoglycemia (Figure 3) indicate that the beneficial effects of MET are independent of food intake.

Microdialysis experiments previously demonstrated that insulin-induced hypoglycemia augments brain dopamine levels.⁴²^,⁴³ A specific role for dopamine in mediating hypoglycemia awareness has previously been suggested by experiments demonstrating that the absence of dopamine action abolishes the food intake responses to hypoglycemia.⁴⁴^,⁴⁵ Consistent with previous reports,³⁴ the current study demonstrates that recurrent episodes of iatrogenic hypoglycemia (Figure 4I) and recurrent dopaminergic agonism (Figure 4N) down-regulate dopaminergic receptor mRNA.

With systemic administration of MET in these experiments, the exact site of MET action cannot be ascertained. However, the fact that the RH induced changes specifically in VTA dopamine D2 receptor and dopamine transporter (SLC6A3) gene expression and that these changes were reversed with MET treatment indicates that MET acted within the CNS. These results are consistent with the notion that (1) HAAF may be mediated (at least in part) by an adaptive response to RH-induced increases in CNS dopamine action and (2) the beneficial effects of the dopaminergic antagonist, MET (in both restoring awareness of hypoglycemia and restoring the CRR to hypoglycemia), may be mediated by the antagonism of these neuroglycopenic-induced increases in CNS dopamine that presumedly occurred during each day of the 3-day recurrent 2DG or RH conditioning period.

Consistent with the beneficial effects of antecedent dopaminergic antagonism, the opposite (i.e., detrimental) effects of antecedent dopaminergic agonism have been reported. Antecedent treatment of normal men with the dopamine receptor agonist, BROMO, blunts the neuroendocrine response to subsequent insulin-induced hypoglycemia.⁵⁰ Consistent with these previous studies, the current study demonstrates that recurrent central BROMO administration, recapitulated the syndrome of HAAF, by both (1) inducing IAH, noted by a blunted food intake response to hypoglycemia (Figure 4C), and (2) virtually abolishing the CRR to hypoglycemia (Figures 4G, 4H, and 4M) with resultant increased glucose infusion rates during the clamp (Figure 4F [∗∗∗∗p < 0.0001 RCSF vs. RBROMO], Figure 4L [∗∗∗p < 0.001 RCSF vs. RBROMO]). These detrimental effects of recurrent dopaminergic agonism were mediated, at least in part, by actions in the VTA (Figures 4K–4M) and were associated with a down-regulation of dopaminergic receptors (Figure 4N). Taken together, these studies are consistent with the notion that RH induces HAAF via RH-induced dopamine release leading to metabolic adaptations consisting of a down-regulation of dopaminergic D2 receptor expression and up-regulation of dopaminergic transporter expression, specifically in the VTA.

Although several dopaminergic areas of the brain were examined in response to RH, key dopamine signaling gene products were only altered in the VTA, and not the VMH, NA, or SN (Figures 4I and S3). The fact that the RH induced changes in DRD2 and SLC6A3 expression in the VTA and that these coordinated changes were reversed with MET treatment is compelling evidence that the beneficial effects of MET are related to, and perhaps mediated by, changes in VTA DRD2 and SLC6A3 expression. Given that D2 receptors exert inhibitory control over dopamine neurons and are the primary target of MET, reduced DRD2 expression following RH may reflect diminished dopaminergic inhibitory tone contributing to IAH and an impaired CRR to hypoglycemia.⁶³^,⁶⁴ Consistent with these findings, administration of BROMO directly into the VTA induced a blunted adrenomedullary response to hypoglycemia that was associated with a downregulation of DRD2 expression (Figures 4M and 4N).

This is the first study, to the authors’ knowledge, to show both improvement in awareness of hypoglycemia and the CRR to hypoglycemia (thereby reversing the two principal components of HAAF) with drug treatment in non-diabetic and STZ-induced diabetic rodent models. Furthermore, the complementary dopamine agonist and antagonist experiments have identified mechanisms that warrant further exploration especially with regard to understanding the mechanism behind altered counterregulation and IAH characteristic of HAAF. In conclusion, these data suggest that MET could become a useful therapeutic for people with diabetes and IAH who are at risk for severe hypoglycemia.

Limitations of the study

Historically, the clinical symptoms of hypoglycemia have been difficult to assess in rodent models. In this study, food intake was used as an objective behavioral proxy for hypoglycemia awareness. Consistent with this approach, acute hypoglycemia consistently increased food intake across multiple experimental conditions (Figures 1A, 1B, 1D, 1E, 1G, 1H, 2F, 2G, 2I, and 2J) and food intake was strongly correlated with decreasing BG levels (Figure 1I), supporting its use as a quantifiable behavioral response to hypoglycemia. With regard to RH preconditioning, as expected, RH blunted the sympathoadrenal response to hypoglycemia (Figures 3H and 3N). However, an important limitation is that RH precondition did not blunt the hypoglycemia-induced increase in food intake (Figures 1E and 1J). Thus, the rodent model of RH does not fully recapitulate both components of clinical HAAF. Therefore, these behavioral and neuroendocrine responses to acute hypoglycemia may be differentially regulated in rodents compared to humans. To address this limitation, R2DG preconditioning was employed as it was previously shown to induce a blunted food intake response.³³ Interestingly, R2DG preconditioning impaired both the food intake response and the sympathoadrenal response to acute insulin-induced hypoglycemia, more closely modeling both components of clinical HAAF (Figures 1H, 1K, 1O, 2G, 2J, 2M, and 3C).

Consistent with other reports, norepinephrine levels did not increase in response to hypoglycemia in this rodent model,⁶⁵ unlike the documented responses in humans.⁶⁶ The measurement of systemic dopamine levels, c-peptide levels, and autonomic nervous system activity represent important goals for future research to better characterize the relationship between dopaminergic signaling and RH with regard to the neuroendocrine responses to hypoglycemia.

I.c.v. administration of BROMO was intentionally used to achieve broad central dopaminergic agonism. Following RH, regional analyses revealed significant alterations in DRD2 and SLC6A3 gene expression selectively within the VTA, providing the rationale for subsequent direct BROMO injections into this region. While direct BROMO injections into the VTA reproduced these changes in gene expression and CRR, we cannot exclude BROMO action in adjacent brain regions.

MET and BROMO are dopamine receptor modulators with high affinity toward D2 receptors.⁶³^,⁶⁴^,⁶⁷ The present data do not exclude possible contributions of other dopamine receptor subtypes. However, recurrent insulin-induced hypoglycemia decreased DRD2 expression and MET reversed this effect (Figure 4I), supporting a dominant role for D2 signaling in this response.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Simon Fisher (simon.fisher@uky.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

•
This study did not generate omics data.
•
This study does not report new code
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

The authors would like to thank and acknowledge research support from DK118082, DK135111, and JDRF (2-SRA-2014-270-M-R) to S.J.F. as well as support from (1) the Diabetes and Metabolism Research Center at the University of Utah, (2) the University of Kentucky Barnstable Brown Diabetes and Obesity Center, and (3) the University of Kentucky Diabetes and Obesity Research Priority Area. M.H.D. was supported by DK109894 and TL1TR001997. We report no conflicts of interest. Parts of this manuscript were presented in abstract format at the American Diabetes Association Scientific Sessions in 2016 (152-OR), 2018 (104-OR), 2019 (118-OR), 2021 (171-OR), and 2022 (164-OR).

Author contributions

Designing research studies, E.M., M.H.D., A.V.d.A., S.S., R.A., O.C., and S.J.F.; conducting experiments, E.M., M.H.D., A.V.d.A., R.A., S.S., P.H., A.M., M.W., E.B., O.C., and S.J.F.; analyzing data, E.M., M.H.D., A.V.d.A., R.A., S.S., P.H., O.C., and S.J.F.; writing the manuscript, E.M., M.H.D., S.S., A.V.d.A., R.A., O.C., and S.J.F.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins

Ketamine	Covetrus	Item #080524
Xylazine	Covetrus	Item #061035
Lidocaine	Covetrus	Item #002468
Rimadyl (carprofen)	Covetrus	Item #083911
Polyvinylpyrrolidone	Sigma	Cas #9003-39-8
Heparin	Fresenius Kabi	C504013
Regular Human Insulin (Humulin R)	Covetrus	Item #067766
Human Insulin Isophane NPH (Humulin-N)	Covetrus	Item #030989
Dextrose solution, 50%	Covetrus	Item #069168
Ethylenediaminetetraacetic acid disodium salt solution	Sigma	Cas #139-33-3
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid	Sigma	Cas #67-45-2
Aprotinin	Sigma	Cas #9087-70-1
Glutathion	Sigma	Cas #70-18-8
Streptozotocin	Sigma	Cas #18883-66-4
Rodent chow (Teklad 2920X)	Inotiv	2920X
2-Deoxy-D-glucose	Sigma	Cas #154-17-6
Fluorojade B	Histo-chem	#2FJB
Metoclopramide	Sigma	Cas #7232-21-5
Bromocriptine mesylate	Sigma	Cas #22260-51-1

Critical commercial assays

Glucagon assay	Millipore	Cat. # GL-32K
Catecholamine assay	Abnova	Catalog #KA1877
miRNeasy Mini Kit	Qiagen	Cat no. 74104
TaqMan Gene Expression Assay (FAM) Drd2	Thermo Fisher	Catalog #4331182 Assay ID RN00561126_m1
TaqMan Gene Expression Assay (FAM) Slc6a3	Thermo Fisher	Catalog #4331182 Assay ID Rn00562224_m1
TaqMan Gene Expression Assay (FAM) Ddc	Thermo Fisher	Catalog #4331182 Assay ID Rn01401189_m1
TaqMan Gene Expression Assay (FAM) Comt	Thermo Fisher	Catalog #4331182 Assay ID Rn00561037
TaqMan Gene Expression Assay (FAM) Actb	Thermo Fisher	Catalog #4331182 Assay ID Rn00667869_m1

Experimental models: Organisms/strains

Sprague-Dawley rats (male)	Charles River Laboratories	Cat#001 RRID: RGD_734476

Software and algorithms

GraphPad Prism	GraphPad Software, LLC	Version 10.5
Microsoft Excel	Microsoft Office	Excel 2019

Other

Micro-Renathane Tubing 0.033 × 0.014	Braintree Scientific Inc.	MRE 033
Micro-Renathane Tubing 0.025 × 0.012	Braintree Scientific	MRE 025
Glucometer (Ascencia Contour, Bayer)	Contour	Manuf #7189
KDS 100 Legacy Syringe Pump	KD Scientific	Code 780100
Lab Standard Stereotaxic Instrument, Rat	Stoelting	Item #51600
Brain Punch Set	Soetling	Item #57401

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Experimental model

Animals

Adult, male Sprague-Dawley rats (weight, 250–300 g; Charles River Laboratories, Malvern, PA) were individually housed in temperature – and light – controlled environments (22°C–24°C with 12-h light/12-h dark cycle). Our study exclusively examined male rats. It is unknown whether the findings are similar for female rats. All studies were done in accordance with and approved by respective IACUC review boards at the University of Utah and the University of Kentucky.

Method details

Vascular surgery

For vessel cannulation, rats were anesthetized with an intraperitoneal (IP) injection of ketamine/xylazine cocktail (75mg/kg Ketamine IP, 5mg/kg Xylazine IP), locally with lidocaine (2-4mg/kg SQ) at both surgical sites, and received 5mg/kg Rimadyl SQ.⁶⁸ A microrenathane catheter (MRE 033, Braintree Scientific Inc.) was inserted into the left carotid artery (for blood sampling), and two catheters (MRE 025, Braintree Scientific Inc.) were implanted into the right jugular vein (for infusions). To retain patency, catheters were filled with 40% polyvinylpyrrolidone (Sigma, St. Louis, MO) in a heparin solution (1,000 Units/mL; USP; Fresenius Kabi USA, LLC).

Hypoglycemic clamps

For the hypoglycemic clamps, after an overnight fast and basal sampling, a constant infusion of insulin (50 mU∗kg⁻¹∗min⁻¹ at 10 μL/min) and a variable infusion of dextrose [40% (BW/vol)] to induce hypoglycemia (BG 40–50 mg/dL) commenced for 90-min.⁵³ For all clamps, arterial BG was measured in 10-min intervals using a glucometer (Ascensia Contour, Bayer HealthCare, IN, USA). Arterial blood samples were collected at times 0, 30, 60 and 90 min, then aliquoted (with appropriate preservatives), and frozen at −80 ֯ C for later determination of catecholamines and glucagon concentrations. Animals were then euthanized at the conclusion of the clamps.

Assessing awareness of hypoglycemia

Food intake is a quantifiable measurement of the response to hypoglycemia in the rodent. Rats were maintained on powdered standard rodent chow (Teklad; irradiated T2920x, Inotiv, USA). For the food intake experiments, chow was weighed prior to placing in individual cages. Rats then received either a subcutaneous (SQ) injection of insulin (NPH 15 U/kg; n = 8) or saline (1 mL/kg SQ; n = 4–7 and had BG values checked at baseline, 2, 4, and 6-h post-injection via glucometer. After 6-h, the chow was measured and food intake was quantified. As part of the drug screen, centrally acting FDA approved drugs were tested to determine if they increased the food intake response in the setting of hypoglycemia.

Development of Impaired Awareness of Hypoglycemia model

To determine if recurrent hypoglycemia preconditioning could induce a model of IAH, rats received either recurrent saline (RS) (1 mL/kg SQ) daily for 3 days or subjected to recurrent insulin induced hypoglycemia (RH; n = 7) daily for 3 days. For this three-day RH preconditioning protocol, insulin (NPH) was dosed at 2.0 U/kg on day 1 and decreased to 1.5, 1.2, respectively on the following days. On day 4, according to the protocol (outlined in Figure 1C), RS rats were treated with a single subcutaneous injection of saline (RS + SAL n = 6) or insulin (15 NPH U/kg; RS + INS n = 6) while RH treated animals were similarly treated with a single subcutaneous injection of insulin (RH + INS, n = 7). Over the course of the next 4–6 h, blood glucose was monitored using a glucometer and food intake was measured. Centrally acting FDA approved drugs were tested to determine if they increased the food intake response in the setting of hypoglycemia.

To create a model of IAH, a separate cohort of rats were either given recurrent 2DG (R2DG; 200 mg/kg/d; Sigma; SQ; n = 10) or recurrent saline (RS 1 mL/kg; RS + SAL n = 3 & RS + INS n = 10) for 3-day followed by, on day 4, a singular insulin injection (15 NPH U/kg SQ) or saline. Blood glucose was measured at baseline, 2, and 4-h post injection and food intake was measured at the end of the experiment.

To determine if R2DG induces brain damage; rats were sacrificed one-week after treatment with either, 1) RS (n = 8), 2) one episode of insulin induced hypoglycemia (NPH; 30 U/kg, 10–15 mg/dL; n = 27), or 3) 3-day of R2DG (200mg/kg/d SQ; n = 12). Brain sections (right cortex, left cortex, and hippocampus) were stained for fluorojade B (Histo-chem), a marker of dead/dying brain cells.⁵³^,⁵⁴^,⁵⁵ To assess whether 2DG induced blunting of the food intake response could be reversed over time, the food intake response to insulin induced hypoglycemia was assessed immediately following three days of preconditioning with SAL (RS + INS, saline 1 mL/kg, NPH 15 U/kg SQ, n = 4) or recurrent 2DG (R2DG + INS, n = 6) or two weeks following the three-day R2DG preconditioning period (two weeks post R2DG + INS, n = 6).

Met increases food intake during hypoglycemia

Metoclopramide was identified as a potential candidate that might augment awareness of hypoglycemia (i.e., increase food intake under hypoglycemic conditions). Briefly, rats were either given saline (n = 6; 1 mL/kg SQ), insulin (n = 6; 15 U/kg SQ), or metoclopramide (3 mg/kg, Sigma, IP) + insulin (n = 6). BG was measured at specified intervals and food intake measured at the end of the experiment.

Treatment of IAH with metoclopramide (MET)

MET and food intake

To determine whether the effects of MET (Sigma Aldrich, USA) to stimulate food intake under hypoglycemic conditions were specific for the stimulus of hypoglycemia and not caused by a non-specific enhancement in food consumption, the effects of MET treatment alone was measured. Rats received a singular injection of either SAL (1 mL/kg SQ; n = 3) or MET (3 mg/kg/d IP; n = 4). BG was measured at specified intervals and food intake measured at the end of the experiment.

Prevention of IAH and restoration of awareness with MET

The ability of MET to treat IAH was assessed under two conditions. The initial series of experiments were to determine if MET could prevent the development of IAH. For this prevention protocol, there were 4 experimental groups (outlined in Figure 2E): 1) RS + SAL (n = 6; 1 mL/kg SQ), 2) RS + INS (n = 6), 3) R2DG + INS (n = 6, 200 mg/kg SQ), and 4) R2DG+MET+INS (n = 8). Thus, preconditioning consisted of treatment with SAL, 2DG, or 2DG + MET as indicated in Figure 2E. MET (3 mg/kg/d IP) was administered one hour prior to insulin induced hypoglycemia during the preconditioning phase (Figure 2E). On day 5, one control group was treated with saline (RS + SAL) while all the other groups received a singular injection (15 NPH U/kg SQ) of insulin to induce hypoglycemia (30–50 mg/dL). MET was not administered on day 5. Blood glucose was measured at baseline, 2, and 4-h and food intake measured at the end of the experiment.

The second series of experiments were designed to determine if MET could restore the hypoglycemia awareness following the induction of IAH. Four experimental groups were assessed (Figure 2G): 1) RS + SAL (n = 8; 1 mL/kg SQ), 2) RS + INS (n = 6), 3) R2DG + INS (n = 6; 200 mg/kg SQ), and 4) R2DG+MET+INS (n = 6; 3 mg/kg/d IP). Following 3 days of recurrent 2DG administration, MET was administered on days 4–6 along with continued R2DG treatment. On day 7, all groups received insulin (15 NPH U/kg SQ). BG was measured at specified intervals and food intake measured at the end of the experiment.

Prevention of IAH with MET treatment in STZ induced diabetic rats

Rats underwent vessel cannulation (described above) and upon recovery were rendered diabetic with a single injection of streptozotocin (STZ; 65 mg/kg IP; Sigma Aldrich, USA). To prepare STZ, 65 mg of STZ was combined with 1 mL of citrate buffer to reach a pH of 4.6. Diabetes was diagnosed as having random blood sugar >250 md/dL. Diabetic rodents were not treated with insulin during the 10-day recovery from cannulation surgery and induction of diabetes. The prevention protocol was carried out with the following experimental groups (Figure 2K): 1) STZ+RS+INS (1 mL/kg SQ; n = 7), 2) STZ+2DG + INS (200 mg/kg SQ; n = 6), 3) STZ+R2DG + MET (3 mg/kg/d; n = 6). Following an overnight fast, all rats had a hyperinsulinemic-hypoglycemic clamp performed and food intake was quantified after 5-h on day 5. During the clamp, animals were required to maintain hypoglycemia for at least 30 min.

Counterregulatory response with and without MET treatment

R2DG model measuring CRR with and without MET treatment

To determine if 2DG would also blunt the glucose CRR and whether or not MET would prevent the blunting of the CRR, three experimental groups were used (Figure 3A): 1) RS + INS (1 mL/kg SQ; n = 9), 2) R2DG + INS (200 mg/kg; SQ; n = 7), and 3) R2DG+MET+INS (10 mg/kg/d IP; n = 6). On day 5, rats underwent a stepped hyperinsulinemic-euglycemic-hypoglycemic clamp, 110 ± 10 mg/dL and 45 ± 5 mg/dL respectively. Blood samples were collected at 0, 30, 60, and 90-min of the clamp. Blood was immediately centrifugated, aliquoted, and frozen for later determination of plasma epinephrine and glucagon concentrations. Blood samples were combined with the appropriate amount of preservatives, ethylene glycol tetraacetic acid (EGTA)-glutathione solution and trasylol (aprotinin), for catecholamines and glucagon, respectively.

Prevention protocol using the RH model measuring CRR with and without MET treatment

Similar to the prevention protocol used in the IAH model, three experimental groups were examined using the RH model (Figure 3E): 1) RS + INS (n = 13), 2) RH + INS (n = 15), and 3) RH+MET+INS (3 mg/kg/d IP; n = 17). Three days prior to RH protocol, rats underwent vessel cannulation surgery (described above). Treatments were with RS, RH, and MET as indicated in Figure 3E. On days 2–4, MET was given to the RH + MET group 1-h prior to the induction of hypoglycemia. RH and RH + MET received a subcutaneous injection of regular insulin (Day 2: 2.0 U/kg, Day 3: 1.5 U/kg, and Day 4: 1.2 U/kg); whereas, the RS group received saline (1 mL/kg SQ). For the RH protocol, the goal was for animals to reach hypoglycemia (45 ± 15 mg/dL) during each consecutive day of the protocol. During RH treatment, BG was monitored with a glucometer in 10-min intervals for 180-min via tail vein nick. On day 5, all rats underwent hyperinsulinemic-hypoglycemic clamp (45 ± 5 mg/dL) and blood samples were taken at −30 min, 0, 60, and 90-min. Samples were centrifugated (with appropriate preservatives), aliquoted, and frozen at – 80 °C.

Restoration protocol using RH model measuring CRR with and without MET treatment

Three experimental groups were used to examine the efficacy of MET on restoring the CRR using the RH model (Figure 3K): 1) RS + INS (n = 7), 2) RH + INS (n = 7), and 3) RH+MET+INS (3 mg/kg/d IP; n = 7). Three days prior to RH protocol, rats underwent vessel cannulation surgery (described above). After recovery from surgery, RH and RH + MET began the RH protocol receiving a subcutaneous injection of insulin days 1–3 (Day 1: 2.0 U/kg, Day 2: 1.5 U/kg, and Day 3: 1.2 U/kg SQ); whereas, the RS group received saline (1 mL/kg SQ) injections. On days 4–6, RH + MET also received intraperitoneal injection of MET 1-h prior to RH. During RH treatment, BG was monitored with a glucometer in 10-min intervals for 180-min via tail vein nick. On day 7, all rats underwent hyperinsulinemic-hypoglycemic (45 ± 5 mg/dL) clamps and blood samples were taken at −30 min, 0, 60, and 90-min. Samples were centrifugated (with appropriate preservatives), aliquoted, and frozen at – 80 °C.

Dopaminergic signaling & the sympathoadrenal response

Recurrent bromocriptine administration in the intracerebral ventricular (ICV) space on food intake

Bromocriptine (BROMO; Sigma) is a dopamine receptor agonist. To determine if recurrent antecedent treatment with BROMO alters the food intake response to acute hypoglycemia (45 ± 5 mg/dL), rats received intracerebroventricular (ICV) injections (from bregma: posterior −2.6mm, medial-lateral 0mm, dorsal-ventral 8.2mm) once daily for 3 days; with either artificial cerebral spinal fluid (aCSF) (1 mL/kg; n = 7) or BROMO (2 μg; n = 7) into the 3^rd ventricle (Figure 4A). ICV injections allow for central delivery and broad distribution of bromocriptine. On day 4, hypoglycemia was induced with a singular injection of insulin (NPH 15 U/kg SQ). BG was measured at specified intervals and food intake measured at the end of the experiment. After each study, brains were sectioned and cannula tip was confirmed to be in the ICV.

Effects of recurrent ICV BROMO administration on the CRR

Rats received 3-day of either aCSF (1 mL/kg; n = 7) or BROMO (2 μg; n = 7) in the ICV (Figure 4D). On day 4, both groups underwent hyperinsulinemic-hypoglycemic clamps (45 ± 5 mg/dL) and blood samples were taken at −30 min, 0, 60, and 90-min. Samples were centrifugated (with appropriate preservatives), aliquoted, and frozen at −80 ֯ C.

Dopaminergic gene expression in different regions of the brain by quantitative real-time PCR (qPCR) with and without MET treatment

To determine the effect of RH on dopaminergic gene expression, rats were treated for 3 days with saline (RS 1 mL/kg SQ; n = 6), recurrent insulin induced hypoglycemia (RH: Day 1: 2.0 U/kg, Day 2: 1.5 U/kg, and Day 3: 1.2 U/kg SQ; n = 7) or insulin induced hypoglycemia with MET treatment (RH + MET: RH conditioning described previously, MET 3 mg/kg/day IP; n = 7). On day 4, in the absence of insulin treatment, animals were sacrificed. Punch biopsies (0.5 mm diameter; Stoelting Co, IL) were taken from frozen brain sections of the substantia nigra, ventral tegmental area (VTA), ventromedial hypothalamus, and nucleus accumbens of all experimental groups. In brief, total RNA was extracted and purified from brain regions using miRNeasy Mini Kit (Qiagen, CA) which was reverse transcribed and then amplified by real-time PCR using Taqman gene assays (Thermo Fisher) for dopamine receptor D2 (Drd2; assay ID: Rn00561126_m1), dopamine transporter or solute carrier family 6, member 3 (Dat or Slc6a3; assay ID: Rn00562224_m1), dopa decarboxylase (Ddc: assay ID: Rn01401189_m1), catechol-O-methyltransferase (Comt; assay ID: Rn00561037_m1) and the housekeeping gene beta-actin (Actb; assay ID: Rn00667869_m1). Samples were run in a 7900 HT standard real-time PCR detection system (Applied Biosystems). The 2-ΔΔCt method was used to calculate the relative differences between experimental and control groups as a fold change in gene expression after normalization with reference to the expression of beta-actin endogenous control.⁶⁹

Recurrent BROMO infusion in VTA effects on CRR and gene expression

After vessel cannulation surgery, rats were mounted in a stereotaxic apparatus (Stoelting Co, IL). A bilateral guide cannula (26-gauge, 1.2 mm, 9.2 mm long) was surgically inserted into the ventral tegmental area (VTA; coordinates from bregma: - 6 mm anteroposterior, ±0.6 mm mediolateral, −8.5 mm dorsoventral) using stereotaxic frame. Three days post-operation, on day 4, rats were randomized to receive one of two treatment (n = 7/group; outlined in Figure 4J): 1) artificial cerebrospinal fluid (RCSF+VTA) or 2) bromocriptine (RBROMO+ VTA; 5 μg/day), into the VTA for 3 consecutive days at a rate of 0.1 μL/min for 10 min, and the cannula was left in the VTA for an additional 5 min to avoid backflow. On day 7, after 3–4 h fasting, hyperinsulinemic hypoglycemic (45 ± 5 mg/dL) clamps were performed in both RCSF and RBROMO groups. Blood samples were collected at baseline and every 30 min after hypoglycemia was reached. Samples were centrifugated (with appropriate preservatives), aliquoted, and frozen at – 80 ֯ C. At the end of clamps, all rats were sacrificed to collect brains to confirm cannula tip placement in the VTA and to assess the gene expression in the VTA by qPCR using Taqman assays as described above.

Hormone analysis

Prior to centrifugation, whole blood was combined with appropriate preservatives (EGTA- Glutathione for catecholamines and aprotinin for glucagon). Blood was centrifugated at 10,000g for 15 s, plasma was aliquoted, and immediately frozen (−20 ֯ C) for later determination of plasma hormone concentrations. Catecholamines (Abnova, Taipei, Taiwan) and glucagon (Millipore, Burlington, MA) were quantified by ELISA and run in duplicate. For epinephrine assay, for plasma samples, the intra-assay CV is 7.3–7.6% as reported by the manufacturer. For the glucagon assay, for plasma samples, the intra-assay CV is 4.4–6.9% as reported by the manufacturer.

Quantification and statistical analysis

Data were analyzed with GraphPad (Prism Inc., USA). Data were normally distributed and did not require transformation. Response variables analyzed in the current study were food intake, glucose infusion rates, brain damage quantification, gene expressions, and blood glucose, epinephrine, and glucagon concentrations. Food intake with explanatory variable of experimental group were analyzed with an analysis of variance (ANOVA). BG concentrations and glucose infusion rates at basal (−30 and 0-min of clamp) and hypoglycemia (90-min of clamp) with explanatory variable of experimental group were analyzed with a repeated measures ANOVA. Brain tissue that had brain damage quantified was analyzed with a one-way ANOVA with explanatory variable of experimental group. Gene expression (i.e., COMT, DDC, DRD2, SLC6A3) was quantified and analyzed with a one-way ANOVA for each brain area (ventral tegmental area, nucleus accumbens, ventromedial hypothalamus, and susbtantia nigra) with explanatory variable of experimental group. Gene expression in the ventral tegmental area with explanatory variable of experimental group (i.e., BROMO treatment or control) was analyzed with an ANOVA. Epinephrine and glucagon concentrations at basal (−30 and 0-min of clamp) and hypoglycemia (90-min of clamp) were analyzed with an ANOVA with explanatory variable of experimental group. If ANOVAs showed statistical significance, a Tukey post-hoc analysis assessed specific differences. Statistical significance was considered at p < 0.05. All data are expressed as mean ± SEM with figures unless otherwise stated in the text.

Published: March 27, 2026

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2026.102710.

Supplemental information

Document S1. Figures S1–S4 and Table S1

mmc1.pdf^{(418.1KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(5MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S4 and Table S1

mmc1.pdf^{(418.1KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(5MB, pdf)}

Data Availability Statement

•
This study did not generate omics data.
•
This study does not report new code
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Role of dopamine in the development of impaired counterregulation and impaired awareness of hypoglycemia

Erica Macon

Micah H Devore

Adriana Vieira de Abreu

Rahul Agrawal

Sunny Sharma

Parker Howe

Ashlee Marksbury

Mason Wooten

Evan Brockman

Owen Chan

Simon J Fisher

Summary

Graphical abstract

Highlights

Introduction

Results

Development of IAH rodent model

Figure 1.

Metoclopramide increases food intake during hypoglycemia

Figure 2.

Metoclopramide both prevents the development of IAH and restores awareness of hypoglycemia in an IAH model

Prevention of a blunted CRR and restoration of the CRR with MET treatment

Figure 3.

Figure 4.

Sympathoadrenal responses to hypoglycemia with and without the dopaminergic agonist, bromocriptine, and the dopaminergic antagonist, metoclopramide

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model

Animals

Method details

Vascular surgery

Hypoglycemic clamps

Assessing awareness of hypoglycemia

Development of Impaired Awareness of Hypoglycemia model

Met increases food intake during hypoglycemia

Treatment of IAH with metoclopramide (MET)

MET and food intake

Prevention of IAH and restoration of awareness with MET

Prevention of IAH with MET treatment in STZ induced diabetic rats

Counterregulatory response with and without MET treatment

R2DG model measuring CRR with and without MET treatment

Prevention protocol using the RH model measuring CRR with and without MET treatment

Restoration protocol using RH model measuring CRR with and without MET treatment

Dopaminergic signaling & the sympathoadrenal response

Recurrent bromocriptine administration in the intracerebral ventricular (ICV) space on food intake

Effects of recurrent ICV BROMO administration on the CRR

Dopaminergic gene expression in different regions of the brain by quantitative real-time PCR (qPCR) with and without MET treatment

Recurrent BROMO infusion in VTA effects on CRR and gene expression

Hormone analysis

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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