Hypoglycemic Clamp Studies Research Articles

Counterregulatory hormone and symptom responses to hypoglycaemia in people with type 1 diabetes, insulin-treated type 2 diabetes or without diabetes: the Hypo-RESOLVE hypoglycaemic clamp study

AimThe sympathetic nervous and hormonal counterregulatory responses to hypoglycaemia differ between people with type 1 and type 2 diabetes and may change along the course of diabetes, but have not been directly compared. We aimed to compare counterregulatory hormone and symptom responses to hypoglycaemia between people with type 1 diabetes, insulin-treated type 2 diabetes and controls without diabetes, using a standardised hyperinsulinaemic-hypoglycaemic clamp.MaterialsWe included 47 people with type 1 diabetes, 15 with insulin-treated type 2 diabetes, and 32 controls without diabetes. Controls were matched according to age and sex to the people with type 1 diabetes or with type 2 diabetes. All participants underwent a hyperinsulinaemic–euglycaemic-(5.2 ± 0.4 mmol/L)-hypoglycaemic-(2.8 ± 0.13 mmol/L)-clamp.ResultsThe glucagon response was lower in people with type 1 diabetes (9.4 ± 0.8 pmol/L, 8.0 [7.0–10.0]) compared to type 2 diabetes (23.7 ± 3.7 pmol/L, 18.0 [12.0–28.0], p < 0.001) and controls (30.6 ± 4.7, 25.5 [17.8–35.8] pmol/L, p < 0.001). The adrenaline response was lower in type 1 diabetes (1.7 ± 0.2, 1.6 [1.3–5.2] nmol/L) compared to type 2 diabetes (3.4 ± 0.7, 2.6 [1.3–5.2] nmol/L, p = 0.001) and controls (2.7 ± 0.4, 2.8 [1.4–3.9] nmol/L, p = 0.012). Growth hormone was lower in people with type 2 diabetes than in type 1 diabetes, at baseline (3.4 ± 1.6 vs 7.7 ± 1.3 mU/L, p = 0.042) and during hypoglycaemia (24.7 ± 7.1 vs 62.4 ± 5.8 mU/L, p = 0.001). People with 1 diabetes had lower overall symptom responses than people with type 2 diabetes (45.3 ± 2.7 vs 58.7 ± 6.4, p = 0.018), driven by a lower neuroglycopenic score (27.4 ± 1.8 vs 36.7 ± 4.2, p = 0.012).ConclusionAcute counterregulatory hormone and symptom responses to experimental hypoglycaemia are lower in people with type 1 diabetes than in those with long-standing insulin-treated type 2 diabetes and controls.

High intensity interval training as a novel treatment for impaired awareness of hypoglycaemia in people with type 1 diabetes (HIT4HYPOS): a randomised parallel-group study

Aims/hypothesisImpaired awareness of hypoglycaemia (IAH) in type 1 diabetes may develop through a process referred to as habituation. Consistent with this, a single bout of high intensity interval exercise as a novel stress stimulus improves counterregulatory responses (CRR) to next-day hypoglycaemia, referred to as dishabituation. This longitudinal pilot study investigated whether 4 weeks of high intensity interval training (HIIT) has sustained effects on counterregulatory and symptom responses to hypoglycaemia in adults with type 1 diabetes and IAH.MethodsHIT4HYPOS was a single-centre, randomised, parallel-group study. Participants were identified using the Scottish Diabetes Research Network (SDRN) and from diabetes outpatient clinics in NHS Tayside, UK. The study took place at the Clinical Research Centre, Ninewells Hospital and Medical School, Dundee, UK. Participants were aged 18–55 years with type 1 diabetes of at least 5 years’ duration and HbA1c levels <75 mmol/mol (<9%). They had IAH confirmed by a Gold score ≥4, modified Clarke score ≥4 or Dose Adjustment For Normal Eating [DAFNE] hypoglycaemia awareness rating of 2 or 3, and/or evidence of recurrent hypoglycaemia on flash glucose monitoring. Participants were randomly allocated using a web-based system to either 4 weeks of real-time continuous glucose monitoring (RT-CGM) or RT-CGM+HIIT. Participants and investigators were not masked to group assignment. The HIIT programme was performed for 20 min on a stationary exercise bike three times a week. Hyperinsulinaemic–hypoglycaemic (2.5 mmol/l) clamp studies with assessment of symptoms, hormones and cognitive function were performed at baseline and after 4 weeks of the study intervention. The predefined primary outcome was the difference in hypoglycaemia-induced adrenaline (epinephrine) responses from baseline following RT-CGM or RT-CGM+HIIT.ResultsEighteen participants (nine men and nine women) with type 1 diabetes (median [IQR] duration 27 [18.75–32] years) and IAH were included, with nine participants randomised to each group. Data from all study participants were included in the analysis. During the 4 week intervention there were no significant mean (SEM) differences between RT-CGM and RT-CGM+HIIT in exposure to level 1 (28 [7] vs 22 [4] episodes, p=0.45) or level 2 (9 [3] vs 4 [1] episodes, p=0.29) hypoglycaemia. The CGM-derived mean glucose level, SD of glucose and glucose management indicator (GMI) did not differ between groups. During the hyperinsulinaemic–hypoglycaemic clamp studies, mean (SEM) change from baseline was greater for the noradrenergic responses (RT-CGM vs RT-CGM+HIIT: −988 [447] vs 514 [732] pmol/l, p=0.02) but not the adrenergic responses (–298 [687] vs 1130 [747] pmol/l, p=0.11) in those participants who had undergone RT-CGM+HIIT. There was a benefit of RT-CGM+HIIT for mean (SEM) change from baseline in the glucagon CRR to hypoglycaemia (RT-CGM vs RT-CGM+HIIT: 1 [4] vs 16 [6] ng/l, p=0.01). Consistent with the hormone response, the mean (SEM) symptomatic response to hypoglycaemia (adjusted for baseline) was greater following RT-CGM+HIIT (RT-CGM vs RT-CGM+HIIT: −4 [2] vs 0 [2], p<0.05).Conclusions/interpretationIn this pilot clinical trial in people with type 1 diabetes and IAH, we found continuing benefits of HIIT for overall hormonal and symptomatic CRR to subsequent hypoglycaemia. Our findings also suggest that HIIT may improve the glucagon response to insulin-induced hypoglycaemia.Trial registrationISRCTN15373978.FundingSir George Alberti Fellowship from Diabetes UK (CMF) and the Juvenile Diabetes Research Foundation.Graphical

Clinical evaluation of a decision support system for glucose infusion in hypoglycaemic clamp experiments.

To provide a preliminary evaluation of the accuracy and safety of Gluclas decision support system suggestions in a hypoglycaemic clamp study. This analysis was performed using data from 32 participants (four groups with different glucose-insulin regulation: post Roux-en-Y gastric bypass with and without postprandial hypoglycaemia syndrome, postsleeve gastrectomy and non-operated controls) undergoing Gluclas-assisted hypoglycaemic clamps (target: 2.5 mmol/L for 20 minutes at 150 minutes after oral glucose ingestion). Gluclas provided glucose infusion rate suggestions upon manual entry of blood glucose values (every 5 minutes), which were either followed or overruled by investigators after critical review. Accuracy and safety were evaluated by mean absolute error (MAE), mean absolute percentage error (MAPE), average glucose level, coefficient of variation (CV) and minimal glucose level during the 20-minute hypoglycaemic period. Investigators accepted 84% of suggestions, with a mean deviation of 30.33 mg/min. During the hypoglycaemic period, the MAE was 0.16 (0.12-0.24) (median [interquartile range]) mmol/L and the MAPE was 6.12% (4.80%-9.29%). CV was 4.90% (3.58%-7.27%), with 5% considered the threshold for sufficient quality. The minimal glucose level was 2.40 (2.30-2.50) mmol/L. Gluclas achieved sufficiently high accuracy with minimal safety risks in a population with differences in glucose-insulin dynamics, underscoring its applicability to various patient groups.

147-OR: Revisiting the Impact of Opioid Receptor Blockade on Hypoglycemia-Associated Autonomic Failure (HAAF)

HAAF, which has been defined as &amp;gt;20% reduction in average epinephrine (Epi) levels following recurrent hypoglycemia (PMID: 28911152), complicates insulin therapy in T1D. We previously showed that activation of opioid receptors induces HAAF, while intravenous (IV) administration of the opioid receptor antagonist naloxone prevents HAAF. Because of its wide clinical use and possible direct CNS effects, we examined the effects of intranasal naloxone (IN) on hypoglycemia and HAAF. Seventeen (15M, age 43.8±8.7) healthy adults were enrolled in a randomized, placebo-controlled, cross-over, double-blinded study in which they underwent three hyperinsulinemic hypoglycemic clamp studies (duration 2h, glucose nadir 54 mg/dL), with administration of IN (4mg) or placebo during the first two clamps. Two subjects were excluded for lack of Epi response to hypoglycemia. Nine of the remaining 15 subjects developed HAAF, confirming inter-individual variability in susceptibility to HAAF (PMID: 32915987, 35475026). Among the 15 subjects included in the analysis, Epi levels trended downward between the 1st and 3rd clamps in placebo studies (mean ± SEM: 677 ± 114 vs. 512 ± 78 pg/mL, p=0.054), whereas Epi levels remained stable in IN studies (1st vs. 3rd: 519 ± 70 vs. 487 ± 77 pg/mL, p= 0.69). Among the 9 subjects who developed HAAF, Epi levels during the 1st clamp were significantly lower with IN vs. placebo (493 ± 102 vs. 858 ± 161 pg/mL, p=0.03), but there was no further reduction in Epi with IN (1st vs. 3rd: 493±102 vs. 539±101 pg/ml, p=0.67). In summary, these studies suggest differential effects of intranasal naloxone on initial vs. repeated hypoglycemic episodes. Specifically, IN may be effective in preventing Epi blunting during repeated exposure to hypoglycemia. In subjects susceptible to HAAF, IN may paradoxically blunt the Epi response to initial hypoglycemic exposure. Future studies should investigate the clinical significance of initial vs. ongoing epinephrine responses to hypoglycemia. Disclosure C.E.Boyle: None. M.Hawkins: None. E.Lontchi yimagou: Employee; Sanofi. S.Aleksic: None. P.M.Mathias: None. M.Nogueira cordeiro: None. A.Manavalan: None. M.Carey: None. N.Jiang: None. O.Sandu: None. I.Gabriely: None. K.Zhang: None. Funding National Institutes of Health (R01DK079974)

The Official Journal of ATTD Advanced Technologies &amp; Treatments for Diabetes Conference 22‐25 February 2023 I Berlin &amp; Online

The Official Journal of ATTD Advanced Technologies &amp; Treatments for Diabetes Conference 22‐25 February 2023 I Berlin &amp; Online

Alteration of circulating platelet-related and diabetes-related microRNAs in individuals with type 2 diabetes mellitus: a stepwise hypoglycaemic clamp study

BackgroundIn patients with type 2 diabetes mellitus (T2DM) an association between severe hypoglycaemic episodes and the risk of cardiovascular (CV) morbidity and mortality has been previously established.MethodsWe aimed to investigate the influence of hypoglycaemia on several diabetes-related and platelet-related miRNAs selected based on bioinformatic analysis and literature search, including hsa-miR-16, hsa-miR-34a, hsa-miR-129-2, hsa-miR-15a, hsa-miR-15b, hsa-miR-106a, miR-223, miR-126. Selected miRNAs were validated by qRT-PCR in 14 patients with T2DM on metformin monotherapy, without established CV disease and antiplatelet therapy during a stepwise hypoglycaemic clamp experiment and a follow-up 7 days after the clamp event. In order to identify which pathways and phenotypes are associated with validated miRNAs we performed target prediction on genes expressed with high confidence in platelets.ResultsCirculating levels of miR-106a-5p, miR-15b, miR-15a, miR-16-5p, miR-223 and miR-126 were increased after euglycaemic clamp followed by hypoglycaemic clamp, each with its distinctive time trend. On the contrary, miR-129-2-3p, miR-92a-3p and miR-34a-3p remained unchanged. MiR-16-5p was negatively correlated with interleukin (IL)-6, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) (p = 0.002, p < 0.001, p = 0.016, respectively), whereas miR-126 was positively correlated with VCAM (p < 0.001). There were negative correlations between miR-16-5p, miR-126 and coagulation factors, including factor VIII and von Willebrand factor (vWF). Among all studied miRNAs, miR-126, miR-129-2-3p and miR-15b showed correlation with platelet function. Bioinformatic analysis of platelet-related targets of analyzed miRNAs showed strong enrichment of IL-2 signaling. We also observed significant enrichment of pathways and diseases related to cancer, CV diseases, hyperglycemia, and neurological diseases.ConclusionsHypoglycaemia can significantly influence the expression of platelet-enriched miRNAs, with a time trend paralleling the time course of platelet activation. This suggests miRNAs could be exploited as biomarkers for platelet activation in response to hypoglycaemia, as they are probably released by platelets upon activation by hypoglycaemic episodes. Should they hold their promise in clinical endpoint studies, platelet-derived miRNAs might become helpful markers of CV risk in subjects with diabetes.Trial registration The study was registered at clinical trials.gov; Impact of Hypoglycaemia in Patients With DIAbetes Mellitus Type 2 on PLATElet Activation (Diaplate), trial number: NCT03460899

Brain Permeable AMP-Activated Protein Kinase Activator R481 Raises Glycaemia by Autonomic Nervous System Activation and Amplifies the Counterregulatory Response to Hypoglycaemia in Rats.

AimWe evaluated the efficacy of a novel brain permeable “metformin-like” AMP-activated protein kinase activator, R481, in regulating glucose homeostasis.Materials and MethodsWe used glucose sensing hypothalamic GT1-7 neuronal cells and pancreatic αTC1.9 α-cells to examine the effect of R481 on AMPK pathway activation and cellular metabolism. Glucose tolerance tests and hyperinsulinemic-euglycemic and hypoglycemic clamps were used in Sprague-Dawley rats to assess insulin sensitivity and hypoglycemia counterregulation, respectively.Results In vitro, we demonstrate that R481 increased AMPK phosphorylation in GT1-7 and αTC1.9 cells. In Sprague-Dawley rats, R481 increased peak glucose levels during a glucose tolerance test, without altering insulin levels or glucose clearance. The effect of R481 to raise peak glucose levels was attenuated by allosteric brain permeable AMPK inhibitor SBI-0206965. This effect was also completely abolished by blockade of the autonomic nervous system using hexamethonium. During hypoglycemic clamp studies, R481 treated animals had a significantly lower glucose infusion rate compared to vehicle treated controls. Peak plasma glucagon levels were significantly higher in R481 treated rats with no change to plasma adrenaline levels. In vitro, R481 did not alter glucagon release from αTC1.9 cells, but increased glycolysis. Non brain permeable AMPK activator R419 enhanced AMPK activity in vitro in neuronal cells but did not alter glucose excursion in vivo.ConclusionsThese data demonstrate that peripheral administration of the brain permeable “metformin-like” AMPK activator R481 increases blood glucose by activation of the autonomic nervous system and amplifies the glucagon response to hypoglycemia in rats. Taken together, our data suggest that R481 amplifies the counterregulatory response to hypoglycemia by a central rather than a direct effect on the pancreatic α-cell. These data provide proof-of-concept that central AMPK could be a target for future drug development for prevention of hypoglycemia in diabetes.

Impact of Hybrid Closed Loop Therapy on Hypoglycemia Awareness in Individuals with Type 1 Diabetes and Impaired Hypoglycemia Awareness.

Objective: This study evaluated the efficacy of using a hybrid closed loop (HCL) system in restoring hypoglycemia awareness in individuals with impaired awareness of hypoglycemia (IAH). Research Design and Methods: Participants with IAH (Gold score ≥4) were recruited into a randomized crossover pilot study. They participated in two 8-week periods using a HCL system (Medtronic 670G™) (intervention) and standard insulin pump therapy (control). Hyperinsulinemic hypoglycemic clamp studies were undertaken at baseline and at the end of each study period for the evaluation of the counter-regulatory hormonal and symptomatic responses to hypoglycemia. Results: Seventeen participants (mean age [standard deviation] 35.8 years [11.2 years]) were included in the study. Peak epinephrine levels (median, interquartile range [IQR]) in response to hypoglycemia were similar postintervention and control periods; 234.7 pmol/L (109.2; 938.9) versus 188.3 pmol/L (133.7; 402.9), P = 0.233. However, both peak adrenergic and neuroglycopenic symptom scores were higher after intervention; 5.0 (4.5; 9.0) versus 4.0 (4.0; 5.5), P = 0.009, and 8.5 (6.0; 15.0) versus 6.5 (6.0; 7.0) P = 0.014, respectively. Self-reported hypoglycemia awareness improved: median (IQR) Gold score was 4.0 (3.0; 5.5) versus 5.5 (4.5; 6.0); intervention versus control, P = 0.033. Time spent <3.9 and <3.0 mmol/L was lower in the intervention group than in control, P = 0.002. Other patient-reported outcomes (hypoglycemia fear and diabetes treatment satisfaction) did not change. Conclusions: A short-term use of a HCL system failed to demonstrate an improvement in counter-regulatory hormonal responses. However, higher hypoglycemia symptom scores during controlled hypoglycemia, better self-reported hypoglycemia awareness, and less time spent in hypoglycemia suggest the potential benefits of a HCL system in people with IAH. Trial Registration: anzctr.org.au Identifier: ACTRN12616000909426.

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.

1767-P: Intranasal Naloxone Prevents the Development of Hypoglycemia-Associated Autonomic Failure (HAAF) in Susceptible Individuals

Repeated hypoglycemic episodes can cause HAAF, i.e., blunted counter-regulatory hormone responses. We have shown that activating opiate receptors can induce HAAF in healthy participants subjected to subsequent experimentally induced hypoglycemia. However, we have noted inter-individual variability in the development of HAAF. In our current study we evaluated the effect of blocking opioid receptors with intranasal naloxone in individuals at risk for HAAF. To identify people susceptible to HAAF, fourteen nondiabetic subjects (13M, age 43.4±8.0, BMI 25.5±2.19 kg/m2) underwent two 120-minute hyperinsulinemic hypoglycemic (nadir 54 mg/dl) clamp studies on Day 1 and a third hypoglycemic clamp study on Day 2. Nine individuals developed HAAF, defined as ≥20% reduction in average epinephrine (EPI) levels (90-120 minutes) between the first and third episodes of hypoglycemia (858 ±161 vs. 494±108 pg/ml, p=0.008). The HAAF-prone subjects underwent similar 3-episode hypoglycemic studies on two subsequent occasions with hourly placebo (randomized, double-blinded) or naloxone (4 mg) on Day 1. While average EPI levels declined from the first to the third hypoglycemic episode by 42% with placebo (p=0.01), levels did not differ with intranasal naloxone (p=0.67). Similarly, plasma glucagon levels decreased significantly from the first to the third hypoglycemic episode with placebo but were maintained with naloxone (Glucagon 1st vs. 3rd episode: Placebo 31.5±8.07 vs. 21.6±5.3 mg/ml, p=0.03; Naloxone 30.5±7.42 vs. 27.0±6.9 mg/ml, p=0.41). Naloxone did not alter plasma cortisol responses or hypoglycemic symptoms. This confirms previous reports of inter-individual variability in development of HAAF. Administration of intranasal naloxone during antecedent hypoglycemia prevented HAAF in susceptible individuals. Inhaled naloxone may offer a feasible approach to ameliorate HAAF in targeted individuals at risk for hypoglycemia-related adverse events. Disclosure A. Manavalan: None. P.M. Mathias: None. S. Aleksic: None. E. Lontchi Yimagou: None. M. Carey: None. J. Moy: None. T. Patel: None. B. Kuo: None. O. Sandu: None. H. Shamoon: None. M. Hawkins: None. Funding National Institutes of Health (R01DK079974)

Detection of Hypoglycemia Using Measures of EEG Complexity in Type 1 Diabetes Patients.

Previous literature has demonstrated that hypoglycemic events in patients with type 1 diabetes (T1D) are associated with measurable scalp electroencephalography (EEG) changes in power spectral density. In the present study, we used a dataset of 19-channel scalp EEG recordings in 34 patients with T1D who underwent a hyperinsulinemic–hypoglycemic clamp study. We found that hypoglycemic events are also characterized by EEG complexity changes that are quantifiable at the single-channel level through empirical conditional and permutation entropy and fractal dimension indices, i.e., the Higuchi index, residuals, and tortuosity. Moreover, we demonstrated that the EEG complexity indices computed in parallel in more than one channel can be used as the input for a neural network aimed at identifying hypoglycemia and euglycemia. The accuracy was about 90%, suggesting that nonlinear indices applied to EEG signals might be useful in revealing hypoglycemic events from EEG recordings in patients with T1D.

113-OR: ADA Presidents' Select Abstract: Dishabituation with High Intensity Exercise Improves Epinephrine Response and Symptomatic Awareness to Hypoglycemia in People with Type 1 Diabetes and Impaired Awareness of Hypoglycemia

Impaired awareness of hypoglycemia (IAH) affects approx. 20-25% of all people with type 1 diabetes (T1D) and is a major risk factor for severe hypoglycemia. We recently proposed that IAH may develop as a form of adaptive memory to repeated hypoglycemia; referred to as Habituation. Consistent with this hypothesis, we demonstrated restoration of defective counterregulatory (CRR) responses to hypoglycemia in a rodent model of IAH following introduction of a novel stress stimulus [high intensity exercise (HIT)]; referred to as Dishabituation. In this study we sought to further test our hypothesis by examining whether a single episode of HIT would amplify counterregulatory response to subsequent hypoglycemia in people with T1D who had IAH (assessed by Gold score ≥4, Modified Clark score≥4 or DAFNE hypoglycaemia awareness rating 2 or 3). We recruited 12 participants to a single-centre, randomized, cross-over study. Individuals were randomized to one episode of HIT (4 x 30 s cycle sprints [2 min recovery] at 150% of max Watts achieved during VO2peak assessment) or rest. The following day they underwent a 90-minute hyperinsulinaemic hypoglycemic clamp study at 2.5 mmol/l with measurement of hormonal CRR and symptom scores. Each clamp study was separated by at least 2-weeks. We found that a single episode of HIT led to a 30% increase in the epinephrine response [mean (SEM) 418.8 +/- 62.84 vs. 541.1 +/- 70.5 pg/ml; p&amp;lt;0.05] accompanied by a significant increase in total symptom scores (Edinburgh Hypoglycemia scale: 24.25 +/- 2.96 vs. 27.5 +/- 3.9, p&amp;lt;0.05) during equivalent hypoglycemia induced the following day. These findings are consistent with the hypothesis that IAH develops in people with T1D as a habituated response and that introduction of a novel stressor can restore, at least partially, the CRR response to hypoglycemia. HIT may represent a novel therapeutic intervention for people with IAH. Disclosure C.M. Farrell: None. D.J. West: Research Support; Self; Arla Foods Ingredients, Dexcom, Inc. S.M. Hapca: None. T. Jones: Research Support; Self; Dexcom, Inc., Medtronic MiniMed, Inc. Speaker's Bureau; Self; Eli Lilly and Company, Roche Diabetes Care. A.D. McNeilly: None. R.J. McCrimmon: Advisory Panel; Self; Eli Lilly and Company, Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; The Leona M. and Harry B. Helmsley Charitable Trust. Funding JDRF; Diabetes UK

116-OR: Hypoglycemia Leads to a Delayed Increase in Platelet and Coagulation Markers in Subjects with Type 2 Diabetes Treated with Metformin Only—Results from an Experimental Stepwise Hypoglycemic Clamp Study

Objective: Although hypoglycemia is associated with increased risk for subsequent cardiovascular events, experimental data investigating the effect of hypoglycemia on coagulation and platelet activation in type 2 diabetes are limited. Research Design and Methods: This monocentric, open, single arm, trial included 14 subjects with type 2 diabetes (10 male/4 female, age 55±7 years, HbA1c 51±7 mmol/mol, diabetes duration 5±4 years) and metformin treatment only. A stepwise hyperinsulinemic hypoglycemic clamp was performed, aiming to investigate parameters of platelet function during predefined plateaus of hypoglycemia (at 63 and 45 mg/dl, for 30 minutes respectively), 24 hours and 7 days later. Additionally, all subjects underwent a hyperinsulinemic euglycemic clamp experiment. Results: While platelet activation assessed by light transmittance aggregometry did not significantly increase following a hypoglycemic clamp, flow cytometry based platelet activation assessment demonstrated a significant increase 24 hours (PAC1CD62PCD63pos p&amp;lt;0.01 compared to baseline, and p&amp;lt;0.001 for PAC1CD63Ppos, respectively) and 7 days after the hypoglycemic clamp (p&amp;lt;0.001 for PAC1CD62Ppos, and PAC1CD63Ppos, and p&amp;lt;0.01 for PAC1CD62PCD63pos in comparison to baseline). In addition, coagulation markers like fibrinogen, PAI-1 and factor VIII also increased, with highest levels 24 hours after the hypoglycemic clamp (p&amp;lt;0.001 for fibrinogen and factor VIII and &amp;lt;0.01 for PAI-1). No platelet or coagulation activation was observed during the hyperinsulinemic, euglycemic clamp. Conclusions: One single event of insulin induced hypoglycemia led to an increase in markers of platelet activation and coagulation, however, the activation occurred with a delay and was evident 24 hours and 7 days after the actual hypoglycemia. Disclosure F. Aberer: None. P.N. Pferschy: None. N.J. Tripolt: None. C. Sourij: None. H. Kojzar: None. B. Prietl: None. S. Kofler: None. M. Brunner: None. R. Riedl: None. E. Novak: None. A.M. Obermayer: None. P. Reitbauer: None. T. Stojakovic: None. F. Aziz: None. A. Oulhaj: None. F. Pruller: None. H. Scharnagl: Research Support; Self; Abbott, Amgen Inc. Speaker's Bureau; Self; Sanofi-Aventis Deutschland GmbH. H. Sourij: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Novo Nordisk A/S, Sanofi-Aventis. Speaker's Bureau; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis.

115-OR: Epinephrine Contributes to Hypoglycemia-Associated Autonomic Failure (HAAF)

Recurrent hypoglycemia leads to HAAF, with blunted counterregulatory hormone responses to subsequent hypoglycemic episodes. A recent report suggested inter-individual differences in susceptibility to HAAF (JCEM 102:3571, 2017). Since adrenergic receptor blockade prevents HAAF (Diabetes 60:602, 2011), we investigated to what extent the rises in plasma epinephrine (EPI) associated with hypoglycemia predict the development of HAAF. Healthy non-diabetic subjects (n=18, age 43±2years, BMI 25±1 kg/m2) underwent two 2-hour hypoglycemic clamp studies (nadir 54mg/dl; 0-2h and 4-6 h) on Day 1 followed by a third 2-hour hypoglycemic clamp on Day 2. Twelve subjects (67%) developed HAAF by the third episode, as defined by at least 20% reductions in peak EPI levels (peak EPI 1st vs. 3rd episode: HAAF subjects 1072±156 vs. 530±87pg/ml, p&amp;lt;0.001). Importantly, peak EPI levels during the 1st hypoglycemic episode were ∼83% higher in the subjects who developed HAAF compared to those who did not (p=0.02). To specifically define the role of EPI in the pathogenesis of HAAF, we challenged an additional seven non-diabetic subjects (age 32±4 years, BMI 25±1 kg/m2) with two 2-hour infusions of EPI (0.03 μg/kg/min;0-2h and 4-6 h) on Day 1 followed by 200-minute stepped hypoglycemic clamps (90, 80, 70 and 60 mg/dl, each for 50 minutes) on Day 2. Compared to saline, EPI infusion on Day 1 induced 40% and 28% reductions in epinephrine response to hypoglycemia at the 70 and 60 mg/dl glucose steps on Day 2, respectively (all p&amp;lt;0.05). There were parallel reductions in hypoglycemic symptoms (all p&amp;lt;0.05). Furthermore, the rate of glucose infusion was higher at all steps after EPI infusion (all p&amp;lt;0.05), consistent with a trend towards lower endogenous glucose production (6,6-D2-glucose). Thus, rises in EPI similar to those seen with hypoglycemia reproduce key features of HAAF in non-diabetic subjects. Marked inter-individual variability in EPI levels in response to hypoglycemia may explain why some people are more prone to develop HAAF. Disclosure E. Lontchi Yimagou: None. S. Aleksic: None. L. Upadhyay: None. S. Sharma: None. M. Carey: None. A. Goyal: None. J. You: None. R. Hulkower: None. S. Murthi: None. W.G. Mitchell: None. H. Shamoon: None. M. Hawkins: Other Relationship; Self; Novo Nordisk Inc. Funding American Diabetes Association (1-18-PMF-024 to E.L.Y.); National Institutes of Health (R01DK079974)

282-OR: Impact of a Hybrid Closed-Loop Insulin Delivery System on Hypoglycemia Awareness in Individuals with Type 1 Diabetes

Background: Individuals with impaired awareness of hypoglycemia (IAH) are at risk of severe hypoglycemia. Although it is known that elimination of hypoglycemia can restore hypoglycemia awareness in this high-risk group, this has been challenging in real life. Technological advancements like closed loop system provide an opportunity to minimize hypoglycemia. Hence, the aim of this study was to evaluate whether a hybrid closed loop insulin delivery system can improve hypoglycemia awareness in individuals with IAH. Methods: Participants with type 1 diabetes and IAH (Gold Score ≥4) were randomized to either a hybrid closed loop system (Medtronic 670G) (intervention) or standard insulin pump therapy (control) for 8 weeks. Gold scores and hyperinsulinemic hypoglycemic clamp studies were undertaken at baseline and at the end of the study with evaluation of the hormonal and symptomatic responses to hypoglycemia. Results: A total of 17 participants completed the study (mean (SD) age 35.8 years (11.2), diabetes duration 24.2 years (11.3), HbA1c 7.8% (1.2)). 8 participants were randomized to control and 9 participants to intervention. The baseline (Median, IQR) Gold score was 6.0 (5.0; 6.0) in the overall cohort and did not differ between groups. At the study end, Gold score improved in the intervention group: control vs. intervention 6.0 (4.5; 6.0) vs. 3.5 (3.0; 4.5); p = 0.027. Symptom scores (adrenergic and neuroglycopenic) during the clamp were not different between the control and intervention groups. Conclusions: The use of a hybrid closed loop system over 8 weeks improved self-reported hypoglycemia awareness, however there was no evidence for improvement in the symptomatic responses to experimentally induced hypoglycemia. Disclosure M. Burckhardt: None. J. Dart: None. G.J. Smith: None. M.B. Abraham: None. N. Paramalingam: None. J.M. O'Dea: None. M. de Bock: None. E.A. Davis: None. T. Jones: Research Support; Self; Dexcom, Inc., Medtronic MiniMed, Inc. Speaker's Bureau; Self; Eli Lilly and Company, Roche Diabetes Care.

Effects of GLP-1 on counter-regulatory responses during hypoglycemia after GBP surgery.

The aim of the study was to explore the role of GLP-1 receptor activation on the counter-regulation and symptoms of hypoglycemia in subjects who have undergone gastric bypass surgery (GBP). Experimental hyperinsulinemic-hypoglycemic clamp study. Twelve post-GBP subjects participated in a randomized cross-over study with two hyperinsulinemic, hypoglycemic clamps (glucose nadir 2.7 mmol/L) performed on separate days with concomitant infusions of the GLP-1 analog exenatide or with saline, respectively. Continuous measurements of metabolites and counter-regulatory hormones as well as assessments of heart rate variability and symptoms of hypoglycemia were performed throughout the clamps. No effect of GLP-1 receptor activation on counter-regulatory hormones (glucagon, catecholamines, cortisol, GH) or glucose infusion rate was seen, but we found indications of a downregulation of the sympathetic relative to the parasympathetic nerve activity, as reflected in heart rate variability. No significant differences in symptom of hypoglycemia were observed. Short-term exposure to a GLP-1 receptor agonist does not seem to impact the counter-regulatory hormonal and metabolic responses in post-GBP subjects during hypoglycemic conditions, suggesting that the improvement in symptomatic hypoglycemia post-GBP seen following treatment with GLP-1 receptor agonists may be mediated by mechanism not directly involved in counter-regulation.

Metabolic and proteomic signatures of hypoglycaemia in type 2 diabetes.

To determine the biochemical changes that underlie hypoglycaemia in a healthy control group and in people with type 2 diabetes (T2D). We report a hypoglycaemic clamp study in seven healthy controls and 10 people with T2D. Blood was withdrawn at four time points: at baseline after an overnight fast; after clamping to euglycaemia at 5 mmol/L; after clamping to hypoglycaemia at 2.8 mmol/L; and 24 hours later, after overnight fast. Deep molecular phenotyping using non-targeted metabolomics and the SomaLogic aptamer-based proteomics platform was performed on collected samples. A total of 955 metabolites and 1125 proteins were identified, with significant alterations in >90 molecules. A number of metabolites significantly increased during hypoglycaemia, but only cortisol, adenosine-3',5'-cyclic monophosphate (cyclic AMP), and pregnenolone sulphate, were independent of insulin. By contrast, identified protein changes were triggered by hypoglycaemia rather than insulin. The T2D group had significantly higher levels of fatty acids including 10-nonadecenoate, linolenate and dihomo-linoleate during hypoglycaemia compared with the control group. Molecules contributing to cardiovascular complications such as fatty-acid-binding protein-3 and pregnenolone sulphate were altered in the participants with T2D during hypoglycaemia. Almost all molecules returned to baseline at 24 hours. The present study provides a comprehensive description of molecular events that are triggered by insulin-induced hypoglycaemia. We identified deregulated pathways in T2D that may play a role in the pathophysiology of hypoglycaemia-induced cardiovascular complications.

GPR119 Agonism Increases Glucagon Secretion During Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein-coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established.

Role of Synaptic Plasticity and EphA5-EphrinA5 Interaction Within the Ventromedial Hypothalamus in Response to Recurrent Hypoglycemia

Hypoglycemia stimulates counterregulatory hormone release to restore euglycemia. This protective response is diminished by recurrent hypoglycemia, limiting the benefits of intensive insulin treatment in patients with diabetes. We previously reported that EphA5 receptor-ephrinA5 interactions within the ventromedial hypothalamus (VMH) influence counterregulatory hormone responses during acute hypoglycemia in nondiabetic rats. In this study, we examined whether recurrent hypoglycemia alters the capacity of the ephrinA5 ligand to activate VMH EphA5 receptors, and if so, whether these changes could contribute to pathogenesis of defective glucose counterregulation in response to a standard hypoglycemic stimulus. The expression of ephrinA5, but not EphA5 receptors within the VMH, was reduced by antecedent recurrent hypoglycemia. In addition, the number of synaptic connections was increased and astroglial synaptic coverage was reduced. Activation of VMH EphA5 receptors via targeted microinjection of ephrinA5-Fc before a hyperinsulinemic hypoglycemic clamp study caused a reduction in the glucose infusion rate in nondiabetic rats exposed to recurrent hypoglycemia. The increase in the counterregulatory response to insulin-induced hypoglycemia was associated with a 150% increase in glucagon release (P < 0.001). These data suggest that changes in ephrinA5/EphA5 interactions and synaptic plasticity within the VMH, a key glucose-sensing region in the brain, may contribute to the impairment in glucagon secretion and counterregulatory responses caused by recurrent hypoglycemia.

β2-Adrenergic receptor agonist administration promotes counter-regulatory responses and recovery from hypoglycaemia in rats

We have previously reported that local activation of β2-adrenergic receptors (B2ARs) in the ventromedial hypothalamus (VMH) enhances hypoglycaemic counter-regulation. This study examines whether peripheral delivery of a selective B2AR agonist could also promote counter-regulatory responses and thereby has potential therapeutic value to limit hypoglycaemia risk. Conscious male Sprague-Dawley rats received an intra-arterial injection of the B2AR specific agonist, formoterol, or a control solution either before a hyperinsulinaemic-hypoglycaemic clamp study or immediately before recovery from insulin-induced hypoglycaemia. In addition, the capacity of a VMH-targeted microinjection of a B2AR antagonist to limit the anti-insulin effect of the B2AR agonist was assessed. Systemic delivery of B2AR agonist markedly reduced the exogenous glucose infusion rate (GIR) required during the hypoglycaemic clamp study. This effect was mediated by blockade of insulin's inhibitory effect on endogenous glucose production. Local blockade of B2ARs within the VMH using a specific antagonist partially diminished the effect of systemic activation of B2ARs during hypoglycaemia at least in part by diminishing the adrenaline (epinephrine) response to hypoglycaemia. Peripheral B2AR agonist injection also enhanced glucose recovery from insulin-induced hypoglycaemia. Systemic B2AR agonist administration acts to limit insulin-induced hypoglycaemia by offsetting insulin's inhibitory effect on hepatic glucose production. This effect appears to be predominately mediated via a direct effect on liver B2ARs, but a small stimulatory effect on B2ARs within the VMH cannot be excluded. Our data suggest that formoterol may have therapeutic value to limit the risk of hypoglycaemia in patients with diabetes.