Hypoglycemia-associated Autonomic Failure Research Articles

TRPC5 controls the adrenaline-mediated counter regulation of hypoglycemia.

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

Effects of Intranasal Naloxone on Hypoglycemia-Associated Autonomic Failure (HAAF) in Susceptible Individuals.

Hypoglycemia-associated autonomic failure (HAAF), defined as blunting of counter-regulatory hormone and symptom responses to recurrent hypoglycemia, remains a therapeutic challenge in diabetes treatment. The opioid system may play a role in HAAF pathogenesis since activation of opioid receptors induces HAAF. Blockade of opioid receptors with intravenous naloxone ameliorates HAAF experimentally, yet is not feasible therapeutically. To investigate the effects of opioid receptor blockade with intranasal naloxone on experimentally-induced HAAF. Randomized, double-blinded, placebo-controlled crossover study. Academic research center. Healthy non-diabetic volunteers. Paired two-day studies, 5-10 weeks apart, each consisting of three consecutive hypoglycemic episodes (hyperinsulinemic hypoglycemic clamps, glucose nadir: 54 mg/dL): two on day 1 with administration of intranasal naloxone vs. placebo, followed by the third episode on day 2. Differences in counter-regulatory hormones responses and hypoglycemia symptoms between first and third hypoglycemic episodes in naloxone vs. placebo studies. Out of 17 participants, 9 developed HAAF, confirming variable inter-individual susceptibility. Among participants susceptible to HAAF, naloxone maintained some hormonal and symptomatic responses to hypoglycemia and prevented the associated requirement for increased glucose infusion. Unexpectedly, naloxone reduced plasma epinephrine and growth hormone responses to the first hypoglycemic episode but prevented further reduction with subsequent hypoglycemia. This is the first study to report that intranasal naloxone, a widely used opioid receptor antagonist, may ameliorate some features of HAAF. Further investigation is warranted into mechanisms of variable inter-individual susceptibility to HAAF and the effects of intranasal naloxone in people with diabetes at risk for HAAF.

Investigating the role of an immediate early gene FOS as a potential regulator of autophagic response to hypoglycemia in embryonic hypothalamic neurons.

Hypoglycemia-associated autonomic failure (HAAF) is a well-established complication of diabetes. Although HAAF has serious outcomes such as recurrent morbidity, coma, and death, the mechanisms of HAAF and its pathological components are largely unknown. Our previous studies have revealed that hypoglycemia is associated with the upregulation of an immediate early gene - FOS. In addition, it is documented that glucose deprivation activates neuronal autophagic activities. Therefore, the present study aimed to identify the role of FOS and one of the core components of the autophagy pathway, Beclin-1 (encoded by the BECN1 gene), in the regulation of autophagic mechanisms in embryonic hypothalamic neurons in response to hypoglycemic conditions. Embryonic Mouse Hypothalamic Cell Line N39 (mHypoE-N39 or N39) was cultured in reduced concentrations of glucose (2000, 900, 500, and 200 mg/L). Gene and protein expression, as well as immunofluorescence studies on autophagy were conducted under different reduced glucose concentrations in N39 hypothalamic neurons with and without FOS and BECN1 gene knockdowns (KD). The outcomes of the present study have demonstrated a significant increase in autophagosome formation and subsequent lysosomal degradation in the hypothalamic neurons in response to reduced glucose concentrations. This hypoglycemic response appears to be lowered to a similar extent in the FOS KD and BECN1 KD cells, albeit insignificantly from the negative control, is indicative of the involvement of FOS in the autophagic response of hypothalamic neurons to hypoglycemia. Moreover, the KD cells exhibited a change in morphology and reduced cell viability compared with the control cells. Our findings suggest that reduced FOS expression could potentially be associated with impaired autophagic activities that are dependent on BECN1, which could lead to decreased or blunted hypothalamic activation in response to hypoglycemia, and this, in turn, may contribute to the development of HAAF.

Hypoglycemia Unawareness-A Review on Pathophysiology and Clinical Implications.

Hypoglycemia is a particular problem in people with diabetes while it can also occur in other clinical circumstances. Hypoglycemia unawareness describes a condition in which autonomic and neuroglycopenic symptoms of hypoglycemia decrease and hence are hardly perceivable. A failure to recognize hypoglycemia in time can lead to unconsciousness, seizure, and even death. The risk factors include intensive glycemic control, prior episodes of severe hypoglycemia, long duration of diabetes, alcohol consumption, exercise, renal failure, and sepsis. The pathophysiological mechanisms are manifold, but mainly concern altered brain glucose sensing, cerebral adaptations, and an impaired hormonal counterregulation with an attenuated release of glucagon, epinephrine, growth hormone, and other hormones, as well as impaired autonomous and neuroglycopenic symptoms. Physiologically, this counterregulatory response causes blood glucose levels to rise. The impaired hormonal counterregulatory response to recurrent hypoglycemia can lead to a vicious cycle of frequent and poorly recognized hypoglycemic episodes. There is a shift in glycemic threshold to trigger hormonal counterregulation, resulting in hypoglycemia-associated autonomic failure and leading to the clinical syndrome of hypoglycemia unawareness. This clinical syndrome represents a particularly great challenge in diabetes treatment and, thus, prevention of hypoglycemia is crucial in diabetes management. This mini-review provides an overview of hypoglycemia and the associated severe complication of impaired hypoglycemia awareness and its symptoms, pathophysiology, risk factors, consequences, as well as therapeutic strategies.

Role of the Adrenal Medulla in Hypoglycaemia-Associated Autonomic Failure-A Diabetic Perspective.

Hypoglycaemia-associated autonomic failure (HAAF) is characterised by an impairment in adrenal medullary and neurogenic symptom responses following episodes of recurrent hypoglycaemia. Here, we review the status quo of research related to the regulatory mechanisms of the adrenal medulla in its response to single and recurrent hypoglycaemia in both diabetic and non-diabetic subjects with particular focus given to catecholamine synthesis, enzymatic activity, and the impact of adrenal medullary peptides. Short-term post-transcriptional modifications, particularly phosphorylation at specific residues of tyrosine hydroxylase (TH), play a key role in the regulation of catecholamine synthesis. While the effects of recurrent hypoglycaemia on catecholamine synthetic enzymes remain inconsistent, long-term changes in TH protein expression suggest species-specific responses. Adrenomedullary peptides such as neuropeptide Y (NPY), galanin, and proenkephalin exhibit altered gene and protein expression in response to hypoglycaemia, suggesting a potential role in the modulation of catecholamine secretion. Of note is NPY, since its antagonism has been shown to prevent reductions in TH protein expression. This review highlights the need for further investigation into the molecular mechanisms involved in the adrenal medullary response to hypoglycaemia. Despite advancements in our understanding of HAAF in non-diabetic rodents, a reliable diabetic rodent model of HAAF remains a challenge.

Current and future therapies to treat impaired awareness of hypoglycemia.

In order to achieve optimal glycemic control, intensive insulin regimes are needed for individuals with Type 1 Diabetes (T1D) and insulin-dependent Type 2 Diabetes (T2D). Unfortunately, intensive glycemic control often results in insulin-induced hypoglycemia. Moreover, recurrent episodes of hypoglycemia result in both the loss of the characteristic warning symptoms associated with hypoglycemia and an attenuated counterregulatory hormone responses. The blunting of warning symptoms is known as impaired awareness of hypoglycemia (IAH). Together, IAH and the loss of the hormonal response is termed hypoglycemia associated autonomic failure (HAAF). IAH is prevalent in up to 25% in people with T1D and up to 10% in people with T2D. IAH and HAAF increase the risk of severe hypoglycemia 6-fold and 25-fold, respectively. To reduce this risk for severe hypoglycemia, multiple different therapeutic approaches are being explored that could improve awareness of hypoglycemia. Current therapies to improve awareness of hypoglycemia include patient education and psychoeducation, the use of novel glycemic control technology, pancreas/islet transplantation, and drug therapy. This review examines both existing therapies and potential therapies that are in pre-clinical testing. Novel treatments that improve awareness of hypoglycemia, via improving the counterregulatory hormone responses or improving hypoglycemic symptom recognition, would also shed light on the possible neurological mechanisms that lead to the development of IAH. To reduce the risk of severe hypoglycemia in people with diabetes, elucidating the mechanism behind IAH, as well as developing targeted therapies is currently an unmet need for those that suffer from IAH.

OR12-05 Blunting Of Sympathetic Activation Of Pancreas As A Cause For Hypoglycemic Unawareness In Hypoglycemia Associated Autonomic Failure

Abstract Disclosure: K. Thakkar: None. R. Hampton: None. M. Jimenez Gonzalez: None. D. Espinoza: None. S. Stanley: None. Introduction: Hypoglycemia activates the autonomic nervous system to suppress pancreatic insulin and increase glucagon release, restoring blood glucose levels to normal. These effects occur partly via sympathetic innervation of pancreatic islets. Recurrent hypoglycemia leads to a blunted autonomic response known as Hypoglycemia Associated Autonomic Failure (HAAF), which further exacerbates hypoglycemia. The mechanisms underlying HAAF are not well-understood, but changes in pancreatic innervation may contribute. Hypothesis: Decreased activity and remodeling of sympathetic innervation to the pancreas contribute to autonomic dysfunction with repeated hypoglycemia. Methods: To examine pancreatic sympathetic innervation with repeated hypoglycemia, C57Bl6 mice were treated with 0, 1, or 5 episodes (0, 1x, or 5x) of insulin-induced hypoglycemia. Pancreata, sympathetic celiac ganglia, and brains were harvested. Pancreatic tissue was immunostained for insulin, glucagon, and tyrosine hydroxylase (TH), a marker for sympathetic innervation. Samples were then cleared using iDISCO+, imaged, and analyzed using Imaris to examine the 3D distribution of innervation and islets. To determine if pancreatic sympathetic nerve function is altered in HAAF, we used viral delivery to express activating chemogenetic constructs specifically in pancreas-projecting sympathetic nerves. We assessed the effects of chemogenetic activation of pancreatic sympathetic innervation during insulin tolerance testing (ITT) in C57Bl6 mice with or without previous recurrent hypoglycemia. Results: 3D image analyses suggest acute hypoglycemia (1x) upregulates islet glucagon staining intensity. By comparison, recurrent hypoglycemia (5x) ​decreases pancreatic TH+ innervation and may blunt hypoglycemia-induced increases in islet glucagon staining intensity. Chemogenetic activation of pancreas sympathetic nerves in mice with repeated hypoglycemia increased blood glucose during an ITT while activation in mice without previous hypoglycemia did not significantly alter blood glucose. These data suggest reduced activity in pancreatic sympathetic innervation with recurrent hypoglycemia that can be restored by chemogenetic activation. Conclusions: Our data suggest repeated episodes of hypoglycemia may remodel the structure and function of peripheral pancreatic sympathetic innervation as compared to one episode of hypoglycemia. Restoring activity in pancreatic sympathetic innervation may partially reverse the severity of hypoglycemia in HAAF. Staining and analysis of celiac ganglia and sympathetic outflow circuits in the CNS will allow determination of the level at which blunting of sympathetic activation and/or remodeling of innervation occurs. Presentation: Friday, June 16, 2023

Ghrelin does not impact the blunted counterregulatory response to recurrent hypoglycemia in mice.

Recurrent episodes of insulin-induced hypoglycemia in patients with diabetes mellitus can result in hypoglycemia-associated autonomic failure (HAAF), which is characterized by a compromised response to hypoglycemia by counterregulatory hormones (counterregulatory response; CRR) and hypoglycemia unawareness. HAAF is a leading cause of morbidity in diabetes and often hinders optimal regulation of blood glucose levels. Yet, the molecular pathways underlying HAAF remain incompletely described. We previously reported that in mice, ghrelin is permissive for the usual CRR to insulin-induced hypoglycemia. Here, we tested the hypothesis that attenuated release of ghrelin both results from HAAF and contributes to HAAF. C57BL/6N mice, ghrelin-knockout (KO) + control mice, and GhIRKO (ghrelin cell-selective insulin receptor knockout) + control mice were randomized to one of three treatment groups: a "Euglycemia" group was injected with saline and remained euglycemic; a 1X hypoglycemia ("1X Hypo") group underwent a single episode of insulin-induced hypoglycemia; a recurrent hypoglycemia ("Recurrent Hypo") group underwent repeated episodes of insulin-induced hypoglycemia over five successive days. Recurrent hypoglycemia exaggerated the reduction in blood glucose (by ~30%) and attenuated the elevations in plasma levels of the CRR hormones glucagon (by 64.5%) and epinephrine (by 52.9%) in C57BL/6N mice compared to a single hypoglycemic episode. Yet, plasma ghrelin was equivalently reduced in "1X Hypo" and "Recurrent Hypo" C57BL/6N mice. Ghrelin-KO mice exhibited neither exaggerated hypoglycemia in response to recurrent hypoglycemia, nor any additional attenuation in CRR hormone levels compared to wild-type littermates. Also, in response to recurrent hypoglycemia, GhIRKO mice exhibited nearly identical blood glucose and plasma CRR hormone levels as littermates with intact insulin receptor expression (floxed-IR mice), despite higher plasma ghrelin in GhIRKO mice. These data suggest that the usual reduction of plasma ghrelin due to insulin-induced hypoglycemia is unaltered by recurrent hypoglycemia and that ghrelin does not impact blood glucose or the blunted CRR hormone responses during recurrent hypoglycemia.

Still Excited, but Less Aroused-The Effects of Nutritional Ketosis on Epinephrine Response and Hypothalamic Orexin Neuron Activation Following Recurrent Hypoglycemia in Diabetic Rats.

Hypoglycemia-associated autonomic failure (HAAF) is a serious, life-threatening complication of intensive insulin therapy, particularly in people with type 1 diabetes. The ketogenic diet is reported to beneficially affect glycemic control in people with type 1 diabetes, however its effects on the neurohormonal counterregulatory response to recurrent hypoglycemia and HAAF development are understudied. In this study we used Sprague Dawley rats to establish a HAAF model under non-diabetic and streptozotocin (STZ)-induced diabetic conditions and determined how nutritional ketosis affected the neurohormonal counterregulation and the activity of energy-sensing orexin (OX) neurons. We found that antecedent hypoglycemia diminished the sympathoexcitatory epinephrine response to subsequent hypoglycemia in chow-fed non-diabetic rats, but this did not occur in STZ-diabetic animals. In all cases a ketogenic diet preserved the epinephrine response. Contrary to expectations, STZ-diabetic keto-fed rats showed reduced OX activity in the recurrent hypoglycemia group, which did not occur in any other group. It is possible that the reduced activation of OX neurons is an adaptation aimed at energy conservation accompanied by diminished arousal and exploratory behaviour. Our data suggests that while a ketogenic diet has beneficial effects on glycemia, and epinephrine response, the reduced activation of OX neurons could be detrimental and warrants further investigation.

Experimentally Induced Hypoglycemia-associated Autonomic Failure in Humans: Determinants, Designs, and Drawbacks.

ContextIatrogenic hypoglycemia remains one of the leading hindrances of optimal glycemic management in insulin-treated diabetes. Recurring hypoglycemia leads to a condition of hypoglycemia-associated autonomic failure (HAAF). HAAF refers to a combination of (i) impaired hormonal counterregulatory responses and (ii) hypoglycemia unawareness to subsequent hypoglycemia, substantially increasing the risk of severe hypoglycemia. Several studies since the 1990s have experimentally induced HAAF, yielding variable results.ObjectiveThe aim of this review was to assess the varying designs, clinical outcomes, potential assets, and drawbacks related to these studies.MethodA systemic literature search was conducted on PubMed and Embase in winter 2021 to include all human studies attempting to experimentally induce HAAF. In different combinations, the search terms used were “hypoglycemia-associated autonomic failure,” “HAAF,” “hypoglycemia,” “recurring,” “recurrent,” “repeated,” “consecutive,” and “unawareness,” yielding 1565 publications. Inclusion criteria were studies that had aimed at experimentally inducing HAAF and measuring outcomes of hormonal counterregulation and awareness of hypoglycemia.ResultsThe literature search yielded 27 eligible publications, of which 20 were successful in inducing HAAF while statistical significantly impairing both hormonal counterregulation and impairing awareness of hypoglycemia to subsequent hypoglycemia. Several factors were of significance as regards inducing HAAF: Foremost, the duration of antecedent hypoglycemia should be at least 90 minutes and blood glucose should be maintained below 3.4 mmol/L. Other important factors to consider are the type of participants, insulin dosage, and the risk of unintended hypoglycemia prior to the study.ConclusionHere we have outlined the most important factors to take into consideration when designing a study aimed at inducing HAAF, including to take into consideration other disease states susceptible to hypoglycemia, thus hopefully clarifying the field and allowing qualified studies in the future.

Hypoglycemic unawareness: challenges, triggers, and recommendations in patients with hypoglycemic unawareness: a case report

BackgroundHypoglycemia is a fairly common complication in diabetic patients, particularly in those on insulin therapy. Hypoglycemia symptoms are classified into two types: autonomic and neuroglycopenic symptoms. If a person develops neuroglycopenic symptoms before the appearance of autonomic symptoms or is asymptomatic until blood sugar levels are very low, the patient will develop hypoglycemic unawareness (HU).Case presentationA 25-year-old Iranian woman with HU presented with a severe hypoglycemic episode. This episode was characterized by loss of consciousness and focal neural deficits, which were unusual symptoms in the patient, who was a medical intern with type 1 diabetes and currently being treated with regular and NPH insulin.ConclusionsHypoglycemia is a common complication in diabetic patients receiving oral or insulin therapy. A patient who is unaware of their condition may experience severe and potentially fatal episodes. These incidents can negatively affect their daily lives as well as their careers and jobs. Hypoglycemia-associated autonomic failure is a possible cause for patients with multiple episodes of severe hypoglycemia. IThe use of a continuous glucose monitoring device with an alarm, if available, can be an excellent option for these patients.

The Stress Hormone ACTH Causes a Prolonged Reduction in Heart Rate Variability

Hypoglycemia is a complex stressor that activates multiple hormonal systems and the sympathetic nervous system. Hypoglycemia also impairs autonomic control of blood pressure. It is possible that stress hormones produced during hypoglycemia (such as ACTH) play a role in the attenuation of autonomic function after recurrent hypoglycemia known as “cardiovascular hypoglycemia‐associated autonomic failure”. Prior work from this placebo‐controlled cross‐over study has shown that ACTH infusion reduces Modified Oxford baroreflex gain and that the reduction persists to the following day. The purpose of this analysis was to determine whether measures of heart rate variability (HRV) demonstrate a similar pattern. In the original study, men and women aged 18 – 55 years (n = 22) completed 2 study visits (ACTH or placebo in random order) with a 1–3‐month washout period between. A study visit consisted of a 3‐day/2‐night inpatient stay with the infusion occurring on day 2. Standard HRV measures from 3 minutes of 0.1 Hz breathing (6 breaths/minute) were collected on each of the 3 days. Overall HRV (time or frequency domain), as well as low frequency HRV, were reduced on the infusion day and the day after by ACTH, with no effect on blood pressure variability. Low‐frequency transfer function gain was similarly reduced, but only on day 3. In addition, these frequency domain gains were significantly correlated with the Modified Oxford derived gains. These results suggests that the ACTH impairment of autonomic control of blood pressure is reflected in HRV measures.

Leptin treatment prevents impaired hypoglycemic counterregulation induced by exposure to severe caloric restriction or exposure to recurrent hypoglycemia.

Hypoglycemia-associated autonomic failure (HAAF) is a maladaptive failure in glucose counterregulation in persons with diabetes (PWD) that is caused by recurrent exposure to hypoglycemia. The adipokine leptin is known to regulate glucose homeostasis, and leptin levels fall following exposure to recurrent hypoglycemia. Yet, little is known regarding how reduced leptin levels influence glucose counterregulation, or if low leptin levels are involved in the development of HAAF. The purpose of this study was to determine the effect of hypoleptinemia on the neuroendocrine responses to hypoglycemia. We utilized two separate experimental paradigms known to induce a hypoleptinemic state: 60% caloric restriction (CR) in mice and three days of recurrent hypoglycemia (3dRH) in rats. A sub-set of animals were also treated with leptin (0.5-1.0μg/g) during the CR or 3dRH periods. Neuroendocrine responses to hypoglycemia were assessed 60min following an IP insulin injection on the terminal day of the paradigms. CR mice displayed defects in hypoglycemic counterregulation, indicated by significantly lower glucagon levels relative to controls, 13.5pmol/L (SD 10.7) versus 64.7pmol/L (SD 45) (p=0.002). 3dRH rats displayed reduced epinephrine levels relative to controls, 1900pg/mL (SD 1052) versus 3670pg/mL (SD 780) (p=0.030). Remarkably, leptin treatment during either paradigm completely reversed this effect by normalizing glucagon levels in CR mice, 78.0pmol/L (SD 47.3) (p=0.764), and epinephrine levels in 3dRH rats, 2910pg/mL (SD 1680) (p=0.522). These findings suggest that hypoleptinemia may be a key signaling event driving the development of HAAF and that leptin treatment may prevent the development of HAAF in PWD.

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.

Repeated Pharmacogenetic Catecholamine Neuron Activation in the Ventrolateral Medulla Attenuates Subsequent Glucoregulatory Responses.

Hindbrain catecholamine (CA) neurons are essential for elicitation of protective counterregulatory responses (CRRs) to glucose deficit, including increased feeding and elevation of circulating corticosterone, epinephrine, and glucose. Severe or repeated antecedent glucoprivation results in attenuation of these CRRs and failure to correct glucose deficit, constituting a potentially lethal condition known as hypoglycemia-associated autonomic failure (HAAF) that may occur in patients with diabetes on insulin therapy. Recently, we demonstrated that selective pharmacogenetic activation of CA neuron subpopulations in the ventrolateral medulla during normoglycemia elicits these CRRs in a site-specific manner. In the present experiment, we examined the effect of repeated pharmacogenetic activation of CA neurons in the A1/C1 cell group on subsequent elicitation of feeding, corticosterone secretion, and respiratory quotient. We found that this prior treatment attenuated these responses to subsequent pharmacogenetic stimulation, similar to attenuation of these CRRs following repeated antecedent glucoprivation. This suggests that functional impairment of A1/C1 CA neurons resulting from antecedent glucoprivation may account, at least in part, for impairment of specific CRRs critical for restoration of normoglycemia in response to glucose deficit. Thus, a pharmacogenetic approach to selective activation of key neural circuits could provide a means of identifying neuropathogenic mechanisms contributing to HAAF.

Plasma Epinephrine Contributes to the Development of Experimental Hypoglycemia-Associated Autonomic Failure.

Recurrent hypoglycemia blunts counter-regulatory responses to subsequent hypoglycemic episodes, a syndrome known as hypoglycemia-associated autonomic failure (HAAF). Since adrenergic receptor blockade has been reported to prevent HAAF, we investigated whether the hypoglycemia-associated rise in plasma epinephrine contributes to pathophysiology and reported interindividual differences in susceptibility to HAAF. To assess the role of hypoglycemia-associated epinephrine responses in the susceptibility to HAAF, 24 adult nondiabetic subjects underwent two 2-hour hyperinsulinemic hypoglycemic clamp studies (nadir 54 mg/dL; 0-2 hours and 4-6 hours) on Day 1, followed by a third identical clamp on Day 2. We challenged an additional 7 subjects with two 2-hour infusions of epinephrine (0.03 μg/kg/min; 0-2 hours and 4-6 hours) vs saline on Day 1 followed by a 200-minute stepped hypoglycemic clamp (90, 80, 70, and 60 mg/dL) on Day 2. Thirteen out of 24 subjects developed HAAF, defined by ≥20% reduction in average epinephrine levels during the final 30 minutes of the third compared with the first hypoglycemic episode (P < 0.001). Average epinephrine levels during the final 30 minutes of the first hypoglycemic episode were 2.3 times higher in subjects who developed HAAF compared with those who did not (P = 0.006).Compared to saline, epinephrine infusion on Day 1 reduced the epinephrine responses by 27% at the 70 and 60 mg/dL glucose steps combined (P = 0.04), with a parallel reduction in hypoglycemic symptoms (P = 0.03) on Day 2. Increases in plasma epinephrine reproduce key features of HAAF in nondiabetic subjects. Marked interindividual variability in epinephrine responses to hypoglycemia may explain an individual's susceptibility to developing HAAF.

Role of the HPA Axis in the Metabolic and Baroreflex Components of Hypoglycemia-Associated Autonomic Failure.

Role of the HPA Axis in the Metabolic and Baroreflex Components of Hypoglycemia-Associated Autonomic Failure.

11-LB: A Phase 2 Study of Liquid Stable Glucagon to Treat Hypoglycemia-Associated Autonomic Failure

Objective: To evaluate a novel, ready-to-use, liquid stable, continuous subcutaneous (SC) glucagon infusion (CSGI; Xeris Pharmaceuticals) for the treatment of Hypoglycemia Associated Autonomic Failure (HAAF) in adults with type 1 diabetes (T1D). Method: This was a Phase 2 double-blind, randomized, placebo-controlled parallel trial in T1D adults with HAAF (Gold Score ≥4). Subjects were randomized to receive 1 month of high-rate CSGI (HCSGI, 80 μg/hour), low-rate CSGI (LCSGI, 20 μg/hour), or placebo. Epinephrine hormone response to hypoglycemia (assessed during stepwise hypoglycemia clamp procedure) and Gold Scores were measured at baseline, end of treatment, and both 3- and 6-months post-treatment. Results: Forty-nine subjects were randomized to receive treatment (HCSGI n=15; LCSGI n=18; placebo n=16). No statistically significant differences in mean epinephrine hormone response or Gold Scores were observed across treatment arms at end of treatment. Long-term follow-up of the subjects was stopped early. For completed follow-up clamps (31 subjects, 3 months; 22 subjects, 6 months), LCSGI did not exhibit differences to placebo. However, HCSGI 3 and 6 month mean epinephrine hormone responses were greater (p=NS) and Gold Scores improved [(-1.1±1.5 vs. 0.5±1.5; 3 months, p=NS) and (-1.2±1.5 vs. -0.6±0.9; 6 months, p=NS)]. The administration of CSGI was associated with mild to moderate nausea (5.9% for LCSGI and, 20% for HCSGI,) and vomiting (11.8% for LCSGI and 13.3% for HCSGI,). CSGI was not associated with pain, injection site reactions, or necrolytic migratory erythema. No related SAEs were reported. Conclusion: Compared to placebo in a small group of T1D adults with HAAF, HCSGI may exhibit improvements in Gold Score at both 3 and 6 months. Mean serum epinephrine may be a suboptimal biomarker of HAAF. CSGI was safely used with concomitant insulin, and there were no related SAEs. Disclosure J. Pettus: Consultant; Self; Diasome Pharmaceuticals, Inc., Lexicon Pharmaceuticals, Inc., MannKind Corporation, Novo Nordisk Inc., Sanofi. S. Nakhle: None. F. Ovalle: Research Support; Self; Gan &amp; Lee Pharmaceuticals, Gilead Sciences, Inc., Lilly Diabetes, Novo Nordisk A/S, Sanofi, Xeris Pharmaceuticals, Inc. M.K. Junaidi: Employee; Self; Xeris Pharmaceuticals, Inc. N.C. Close: Employee; Self; EmpiriStat, Inc. V. Conoscenti: None. A. Nguyen: None.

13-LB: Leptin Treatment Prevents Impaired Hypoglycemic Counterregulation Induced by Exposure to Recurrent Hypoglycemia or Caloric Restriction

Background: Hypoglycemia-associated autonomic failure (HAAF) is a significant barrier to achieving optimal glucose levels in persons with diabetes. Our group and others have demonstrated that low leptin levels are concurrent with impaired hypoglycemic counter-regulation in two separate animal models of HAAF: six days of 60% caloric restriction (CR) in mice and 3 days of recurrent hypoglycemia (3dRH) in rats. In this study, we tested whether leptin treatment during CR or 3dRH could prevent the development of HAAF. Methods: In experiment one, 3 groups of mice (two CR groups and one ad-lib fed control group) received either twice daily leptin (0.5-1μg/g, IP) or PBS. In experiment two, 3 groups of rats (two 3dRH and one control) received daily leptin or PBS injections (0.3-0 .7 mg/kg, IP). One day following the CR or 3dRH paradigms, all animals underwent a hypoglycemic ITT and levels of glucagon, leptin, epinephrine (rats), and corticosterone were assessed 60 min following insulin treatment. Results: As expected, PBS treated CR mice and 3dRH rats had significantly reduced glucagon levels and epinephrine levels relative to controls (24 ± 5 vs. 95 ± 20 pmol/L and 57 ± 12 vs. 110 ± 9 ng/mL, respectively). In contrast, there was no differences in the glucagon levels of leptin treated CR mice and epinephrine levels of leptin treated 3dRH rats relative to their respective control groups. Leptin levels in both CR groups were reduced relative to controls (0.9 ± 0.3 and 0.2 ± 0.1 vs. 3.0 ± 0.5 ng/mL, respectively), while corticosterone was unchanged. Glucagon, corticosterone, and leptin levels were similar across groups in the 3dRH paradigm. Discussion: Utilizing two separate animal models of HAAF, we established that leptin treatment can prevent the loss of hypoglycemic counter-regulation. Future studies investigating whether combining leptin treatment with insulin therapy could significantly reduce hypoglycemic complications in diabetes patients are warranted. Disclosure D. McDougal: None. M. DuVall: None. H.X. Sikaffy: None. R. Jariwala: None. C. Morrison: None. C.M. Hill: None. Funding American Diabetes Association (1-15-JF-37 to D.M.); National Institutes of Health (U54GM104940)

MON-LB110 A Phase 2 Evaluation of a Ready-To-Use Liquid Stable Continuous Subcutaneous Glucagon Infusion for the Treatment of Hypoglycemia Associated Autonomic Failure

OBJECTIVE:A novel, ready-to-use, liquid stable, continuous subcutaneous glucagon infusion (CSGI; Xeris Pharmaceuticals) was evaluated for the treatment of adults with type 1 diabetes (T1D) with documented Hypoglycemia Associated Autonomic Failure (HAAF). METHOD:This was a Phase 2 prospective, multi-center, randomized, placebo-controlled, double-blind, parallel trial in T1D adults with documented HAAF, defined as Gold Scale Score ≥4. Subjects were randomized in a 1:1:1 ratio to receive 4 weeks of continuous treatment (via Omnipod pump) with high rate CSGI (80mcg/hour), low rate CSGI, (20mcg/hour) or placebo (matched infusion rates for low and high rate CSGI). The primary endpoint evaluated at 4 weeks was the percent change of the epinephrine hormone response after 30 minutes of induced hypoglycemia. Epinephrine levels and hypoglycemia symptoms were recorded following a stepwise hypoglycemia clamp, and results compared between study treatment arms. Following the first four weeks, subjects continued for an additional 24 weeks to assess their epinephrine hormone response to hypoglycemia measured at 3 months post-treatment (16 weeks), and 6 months post-treatment (28 weeks). RESULTS:Forty-nine subjects were randomized to receive treatment with high rate CSGI (n=15), low rate CSGI (n=18), or matching placebo (n=16). At the end of study treatment, there were no statistically significant differences between the treatment arms for percent change of epinephrine hormone response during a stepwise hypoglycemia clamp (CSGI vs. placebo; p=0.160). As a result, long-term follow-up of the subjects was stopped early. The long-term follow-up data collected to date will be reported separately. The administration of low and high rate CSGI was associated with mild to moderate nausea (5.9%, 20%, respectively) and vomiting (11.8%, 13.3%, respectively) that was self-limited and required no additional intervention. In this 4-week assessment, CSGI was not associated with pain nor significant injection site reactions, and no SAEs related to CSGI were reported. CONCLUSION:In this proof-of-concept study, CSGI did not demonstrate statistically significant improvements in plasma epinephrine concentration when compared to placebo at 4 weeks. While there were positive epinephrine and improved hypoglycemia awareness responses observed in some subjects, the equivocal efficacy results observed may be explained by the unanticipated and incomplete elimination of hypoglycemia on CSGI therapy, and those on placebo therapy experienced hypoglycemia improvement. CSGI can be safely used with concomitant diabetes medications, and there were no SAEs related to CSGI. The 4-week administration of both low and high rate CSGI was safe and well tolerated.