Hyperinsulinemic Hypoglycemic Clamp Research Articles

166-OR: Brain Responses to Hypoglycemia following Contrasting Hypoglycemia Awareness Restoration Strategies: A Comparison of the HARPdoc and HypoAware Studies

Background: Restoration of symptoms in response to hypogycemia has been seen in people with type 1 diabetes (T1D) and impaired awareness of hypoglycaemia (IAH) using an education and diabetes technology approach (HypoAware Study) and a psycho-educational approach addressing cognitions (HARPdoc Trial) . We investigated regional brain responses to hypoglycemia following restoration of IAH between the interventions. Methods: Twelve people with T1D and IAH from HypoAware and from HARPdoc underwent two-step hyperinsulinemic hypoglycemic clamps before and after their intervention. pCASL fMRI brain blood flow images were acquired. A 2x2 study by timepoint ANOVA interaction design was conducted to compare the effect of each intervention on hypoglycemia-euglycemia difference brain blood flow maps. Within group analysis of the difference maps was conducted using paired t-tests. Results: HARPdoc participants were older, mean±SD age 54.1±12.3 vs. 34.7±9.0 yrs (p<0.001) with similar HbA1c, 7.8±0.9 vs. 7.8±0.7%, Gold score, 6.0±1.0 vs. 6.0±1.0, BMI 24.6±4.6 vs. 24.4±3.6 and diabetes duration 22.2±7.2 vs. 23.6±7.7 yrs (all p>0.05) . Each intervention was associated with enhanced activation in the anterior cingulate cortex (ACC) in within group studies (HARPdoc FWEp=0.048, HypoAware FWEp=0.011) . In the between group analysis, a superior frontal gyrus (SFG) region was more activated at hypoglycemia following HARPdoc than HypoAware (FWEp=0.044) . Conclusion: We have shown different brain responses to hypoglycemia in treated IAH depending on the therapeutic approach. Enhanced ACC responses were expected following recovery of symptoms. However, the cognitive approach of HARPdoc enhances the hypoglycemia response in the SFG, known to be involved in self-awareness and appreciation of symptoms, is unique to HARPdoc and is consistent with its psychological and cognitive nature. This may increase the potential for lasting effect. Disclosure P.Jacob: None. S.A.Amiel: Advisory Panel; Medtronic, Novo Nordisk, Other Relationship; Sanofi. M.Nwokolo: None. P.Choudhary: Advisory Panel; Abbott Diabetes, Lilly Diabetes, Medtronic, Research Support; Novo Nordisk, Speaker's Bureau; Dexcom, Inc., Glooko, Inc., Insulet Corporation, Sanofi. Funding Juvenile Diabetes Research Foundation (3-SRA-2017-484-S-B)

374-P: Identifying the Role and Mechanism by Which GDF15 Enhances the Counterregulatory Response to Hypoglycemia

Severe hypoglycemia is the leading acute mortality risk in patients with diabetes. The counterregulatory gluconeogenic and glycogenolytic response to hypoglycemia is the primary adaptive mechanism to enable survival. To better understand the hormonal response that orchestrates hypoglycemia counterregulation, we employed the insulin tolerance test (ITT) and hyperinsulinemic-hypoglycemic clamp to mimic the two common settings in which hypoglycemia can occur in patients: postprandial insulin overdose and elevated basal insulin infusion, respectively. We found that Growth Differentiation Factor 15 (GDF15) can be induced (33.5±2.1 vs. 172.4±29.2 pg/ml, P<0.in ITT and 46.2±11.6 vs. 2865±547.3 pg/ml, P<0.0in the clamp) . GDF15 mRNA and protein are upregulated in kidney, and the GDF15 tdTomato reporter mouse demonstrates the expression of GDF15 in the S3 segment of the proximal tubule. GDF15 increases hepatic gluconeogenesis (26.1±2.2 vs. 33.5±1.6 mg/kg/min, P<0.05) by increasing intrahepatic lipolysis in a beta-adrenergic receptor-2 (Adrb2) -dependent manner. To investigate the role of GDF15 counterregulation in diabetes, we utilized a streptozotocin (STZ) -induced type 1 diabetes (T1D) model. Mice with T1D exhibit impaired GDF15 production in an ITT. Additionally, we found that recurrent hypoglycemia impairs endogenous glucose production during hypoglycemia in mice (27.5±4 vs. 10.5±1.3 mg/kg/min, P<0.01) , which may result from lower GDF15 production (1372±248.2 vs. 648.5±71.3 pg/ml, P<0.05) . In humans, hypoglycemic clamps in healthy volunteers can induce GDF15 production (p=0.01) , although this induction was absent in patients with T1D. Conclusion: These data show the kidney can produce GDF15 in response to hypoglycemia, thereby increasing hepatic gluconeogenesis to enable survival. However, T1D patients are unable to initiate a functional GDF15 signaling pathway resulting in an impaired glucose counterregulatory response to hypoglycemia. Disclosure Z.Li: None. C.Zhang: None. R.Chang: None. J.J.Hwang: None. A.Wang: Research Support; NGM Biopharmaceuticals. R.J.Perry: None. X.Zhang: None. W.Zhu: None. Q.Xu: None. C.Yu: None. B.G.Costa lima: None. X.Li: None. K.Israni-winger: None. A.Nasiri: None. Funding National Institutes of Health (P30 DK045735) National Institutes of Health (R37 CA258271-01A1)

Impact of Polymorphism in the β2-Receptor Gene on Metabolic Responses to Repeated Hypoglycemia in Healthy Humans.

The Arg16 variant in the β2-receptor gene is associated with increased risk of severe hypoglycemia in subjects with type 1 diabetes mellitus. We hypothesized that the Arg16 variant is associated with decreased metabolic and symptomatic responses to recurrent hypoglycemia. Twenty-five healthy male subjects selected according to ADRB2 genotype and being homozygous for either Arg16 (AA; n = 13) or Gly16 (GG; n = 12) participated in 2 consecutive trial days with 3 periods of hypoglycemia (H1-H3) induced by a hyperinsulinemic hypoglycemic clamp. The main outcome measure was mean glucose infusion rate (GIR) during H1-H3. During H1-H3, there was no difference between AA or GG subjects in GIR, counter-regulatory hormones (glucagon, epinephrine, cortisol, growth hormone), or substrate levels of lactate, glycerol, and free fatty acids (FFAs), and no differences in symptom response score or cognitive performance (trail making test, Stroop test). At H3, lactate response was reduced in both genotype groups, but AA subjects had decreased response (mean ± standard error of the mean of area under the curve) of glycerol (-13.1 ± 3.8 μmol L-1 hours; P = .0052), FFA (-30.2 ± 11.1 μmol L-1 hours; P = .021), and β-hydroxybutyrate (-0.008 ± 0.003 mmol L-1 hour; P = .027), while in GG subjects alanine response was increased (negative response values) (-53.9 ± 20.6 μmol L-1 hour; P = .024). There was no difference in GIR between genotype groups, but secondary outcomes suggest a downregulation of the lipolytic and β-hydroxybutyrate responses to recurrent hypoglycemia in AA subjects, in contrast to the responses in GG subjects.

Altered functional connectivity during hypoglycaemia in type 1 diabetes

Behavioural responses to hypoglycaemia require coordinated recruitment of broadly distributed networks of interacting brain regions. We investigated hypoglycaemia-related changes in brain connectivity in people without diabetes (ND) and with type 1 diabetes with normal (NAH) or impaired (IAH) hypoglycaemia awareness. Two-step hyperinsulinaemic hypoglycaemic clamps were performed in 14 ND, 15 NAH and 22 IAH participants. BOLD timeseries were acquired at euglycaemia (5.0 mmol/L) and hypoglycaemia (2.6 mmol/L), with symptom and counter-regulatory hormone measurements. We investigated hypoglycaemia-related connectivity changes using established seed regions for the default mode (DMN), salience (SN) and central executive (CEN) networks and regions whose activity is modulated by hypoglycaemia: the thalamus and right inferior frontal gyrus (RIFG). Hypoglycaemia-induced changes in the DMN, SN and CEN were evident in NAH (all p < 0.05), with no changes in ND or IAH. However, in IAH there was a reduction in connectivity between regions within the RIFG (p = 0.001), not evident in the ND or NAH groups. We conclude that hypoglycaemia induces coordinated recruitment of the DMN and SN in diabetes with preserved hypoglycaemia awareness which is absent in IAH and ND. Changes in connectivity in the RIFG, a region associated with attentional modulation, may be key in impaired hypoglycaemia awareness.

N-Hydroxyethyl-1-Deoxynojirimycin (Miglitol) Restores the Counterregulatory Response to Hypoglycemia Following Antecedent Hypoglycemia.

Antecedent hypoglycemia suppresses the counterregulatory responses to subsequent hypoglycemic episodes, which can be prevented by normalizing portal-mesenteric vein (PMV) glycemia alone during the antecedent bout. Since the sodium-glucose transporter 3 receptor has been implicated in PMV glucosensing, we hypothesized that PMV infusion of the sodium-glucose cotransporter 3 receptor agonist N-hydroxyethyl-1-deoxynojirimycin (miglitol) would rescue the sympathoadrenal response to subsequent hypoglycemia. Rats underwent hyperinsulinemic-hypoglycemic clamps on 2 consecutive days without miglitol infusion (antecedent hypoglycemia without miglitol [HYPO]) or with miglitol infused upstream in the PMV, perfusing the glucosensors, or adjacent to the liver, bypassing PMV glucosensors, on day 1 or day 2. Control animals underwent day 1 euglycemic clamps, followed by hypoglycemic clamps on day 2. Peak epinephrine (EPI) responses for HYPO on day 2 were significantly blunted when compared with controls. Miglitol infusion on day 1 proved ineffective in restoring the EPI response following antecedent hypoglycemia, but day 2 miglitol infusion restored EPI responses to control levels. As norepinephrine and glucagon demonstrated similar responses, day 2 administration of miglitol effectively restored the counterregulatory response following antecedent hypoglycemia. In subsequent experiments, we demonstrate similar results with reduced miglitol infusion doses, approaching those currently prescribed for type 2 diabetes (correcting for rodent size), as well as the efficacy of oral miglitol administration in restoring the counterregulatory responses following antecedent hypoglycemia.

SGLT2 Inhibition Increases Fasting Glucagon but Does Not Restore the Counterregulatory Hormone Response to Hypoglycemia in Participants With Type 1 Diabetes.

Individuals with type 1 diabetes have an impaired glucagon counterregulatory response to hypoglycemia. Sodium-glucose cotransporter (SGLT) inhibitors increase glucagon concentrations. We evaluated whether SGLT inhibition restores the glucagon counterregulatory hormone response to hypoglycemia. Adults with type 1 diabetes (n = 22) were treated with the SGLT2 inhibitor dapagliflozin (5 mg daily) or placebo for 4 weeks in a randomized, double-blind, crossover study. After each treatment phase, participants underwent a hyperinsulinemic-hypoglycemic clamp. Basal glucagon concentrations were 32% higher following dapagliflozin versus placebo, with a median within-participant difference of 2.75 pg/mL (95% CI 1.38-12.6). However, increased basal glucagon levels did not correlate with decreased rates of hypoglycemia and thus do not appear to be protective in avoiding hypoglycemia. During hypoglycemic clamp, SGLT2 inhibition did not change counterregulatory hormone concentrations, time to recovery from hypoglycemia, hypoglycemia symptoms, or cognitive function. Thus, despite raising basal glucagon concentrations, SGLT inhibitor treatment did not restore the impaired glucagon response to hypoglycemia. We propose that clinical reduction in hypoglycemia associated with these agents is a result of changes in diabetes care (e.g., lower insulin doses or improved glycemic variability) as opposed to a direct, physiologic effect of these medications on α-cell function.

Ghrelin cell-expressed insulin receptors mediate meal- and obesity-induced declines in plasma ghrelin.

Mechanisms underlying postprandial and obesity-associated plasma ghrelin reductions are incompletely understood. Here, using ghrelin cell–selective insulin receptor–KO (GhIRKO) mice, we tested the impact of insulin, acting via ghrelin cell–expressed insulin receptors (IRs), to suppress ghrelin secretion. Insulin reduced ghrelin secretion from cultured gastric mucosal cells of control mice but not from those of GhIRKO mice. Acute insulin challenge and insulin infusion during both hyperinsulinemic-hypoglycemic clamps and hyperinsulinemic-euglycemic clamps lowered plasma ghrelin in control mice but not GhIRKO mice. Thus, ghrelin cell–expressed IRs are required for insulin-mediated reductions in plasma ghrelin. Furthermore, interventions that naturally raise insulin (glucose gavage, refeeding following fasting, and chronic high-fat diet) also lowered plasma ghrelin only in control mice — not GhIRKO mice. Thus, meal- and obesity-associated increases in insulin, acting via ghrelin cell–expressed IRs, represent a major, direct negative modulator of ghrelin secretion in vivo, as opposed to ingested or metabolized macronutrients. Refed GhIRKO mice exhibited reduced plasma insulin, highlighting ghrelin’s actions to inhibit insulin release via a feedback loop. Moreover, GhIRKO mice required reduced glucose infusion rates during hyperinsulinemic-hypoglycemic clamps, suggesting that suppressed ghrelin release resulting from direct insulin action on ghrelin cells usually limits ghrelin’s full potential to protect against insulin-induced hypoglycemia.

171-OR: Dopaminergic Signaling in the Ventral Tegmental Area of the Brain Mediates the Impaired Sympathoadrenal Response to Hypoglycemia

The mechanism by which recurrent hypoglycemia (RH) impairs the sympathoadrenal response to hypoglycemia remains unknown. To explore altered brain dopaminergic signaling as a mechanism, 10 weeks old rats were randomized to 3 days: 1) recurrent saline (RS), 2) insulin (2-1.2 U/kg, sc) induced RH (25-50 mg/dl), or 3) RH + antecedent dopamine receptor antagonist metoclopramide (MET, 3 mg/kg, ip). qPCR of brain biopsies demonstrated RH downregulated dopaminergic signaling, noted by decreased expression of dopamine D2 receptor (Drd2, 52%*) and increased dopamine transporter (DAT, ~1.9-fold*) in the ventral tegmental area (VTA); an effect prevented by MET (Figure 1A). To test whether recurrent dopamine activation in the VTA is sufficient to impair the sympathoadrenal response to hypoglycemia, a second rat cohort was randomized to 3-day recurrent VTA infusion of: 1) aCSF (control), or 2) Drd2 agonist bromocriptine (Bromo; 5 µg/d). During hyperinsulinemic (20 mU/kg/min) hypoglycemic (∼45 mg/dl) clamps, recurrent Bromo resulted in an increased glucose infusion rate (1.7-fold*) and blunted epinephrine response (~50%*, Figure 1B). These results demonstrate that blunted dopaminergic signaling in the VTA induced by RH contributes to the impaired sympathoadrenal response, which may be associated with recurrent brain dopaminergic activation. *p<0.05.View largeDownload slideView largeDownload slide DisclosureR. Agrawal: None. S. Sharma: None. S. Fisher: None. FundingNational Institutes of Health (R01NS070235-01A1, R01DK118082)

48-OR: Changes in Global Cerebral Blood Flow in Response to Hypoglycemia in Type 1 Diabetes Do Not Influence Awareness Status

Changes in global cerebral blood flow (gCBF) have been suggested as a mechanism for mitigating the functional impact of hypoglycemia on the brain. The impact of type 1 diabetes (T1D) or hypoglycemia awareness status is not clear. We measured gCBF during progressive lowering of plasma glucose in people with and without T1D, and with retained or impaired awareness of hypoglycemia. Methods: Fifty-one participants (15 with no diabetes (ND), 15 with T1D and normal awareness of hypoglycemia (NAH) and 21 with T1D and impaired awareness of hypoglycemia (IAH)) underwent a two-step hyperinsulinemic hypoglycemic clamp. gCBF was estimated using pseudocontinuous arterial spin labelling magnetic resonance imaging at 6 timepoints: 2 euglycemic, two in the descent phase and two hypoglycemic. gCBF was extracted using a global grey matter mask in SPM12. Statistical analysis and thresholded linear models were performed in R. Results: The mean (+ SD) glucose values at the six timepoints were 95.8 (8.0), 95.5 (9.2), 55.0 (8.3), 50.8 (5.3), 45.7 (4.3) and 45.8 (4.0) mg/dL. Symptom responses to hypoglycemia were absent in IAH. There was a significant increase in CBF in response to hypoglycemia (3-way ANOVA p = 0.04). This was driven by responses in NAH (+7.3%, p &amp;lt; 0.05) and IAH (+10.6% p &amp;lt; 0.01) with no response in ND (+3.5% p = 0.16). The response was not linear, with glucose thresholds of 55.8 mg/dL in NAH and 54.0 mg/dL in IAH (pNAH vs IAH = 0.9) with no threshold in ND. Conclusion: In people with T1D, there is a non-linear relationship between falling glucose and rising gCBF which is not related to awareness status and is not seen in health. The threshold glucose between 54 and 56 mg/dL corresponds closely with level 2 hypoglycemia cut-offs used in current clinical practice. We propose that there is a failure of cerebral vascular autoregulation in response to hypoglycemia in T1D. gCBF responses to hypoglycemia in T1D are independent of symptomatic responses and not responsible for lack of hypoglycemia awareness. Disclosure P. Jacob: None. O. Odaly: None. S. A. Amiel: Advisory Panel; Self; Diabetes UK, Medtronic, Other Relationship; Self; Sanofi, Research Support; Self; Diabetes UK, European Union, JDRF. P. Choudhary: Advisory Panel; Self; Abbott Diabetes, Insulet Corporation, Lilly Diabetes, Medtronic, Sanofi-Aventis, Speaker’s Bureau; Self; Dexcom, Inc., Novo Nordisk. Funding JDRF (3-SRA-2017-484-S-B)

Hypoglycemia Impairs Baroreflex Sensitivity: Implications for Autonomic Control of Cardiovascular Function in Diabetes

Background: Iatrogenic hypoglycemia is an unintended, though common, occurrence in individuals with diabetes. There is a clear association between hypoglycemia exposure and an increase in mortality in individuals with type 2 diabetes (T2DM). It is well-established that recurrent hypoglycemic episodes impair the counterregulatory hormone responses; however, it is yet to be determined if in T2DM there are more global effects of hypoglycemia on autonomic control of cardiovascular function (baroreflex sensitivity, BRS), as has been shown in healthy individuals. This is a clinically relevant knowledge gap as decreases in BRS are pro-arrhythmogenic and associated with decreases in mortality in individuals with diabetes. Objective: We tested the hypothesis that in individuals with T2DM, hypoglycemia impairs baroreflex sensitivity (BRS), a robust measure of cardiovascular autonomic control. Methods: Individuals with well-controlled T2DM and without known cardiovascular disease were exposed to two 90-minute episodes of experimental hypoglycemia (50 mg/dl) in the same day. All individuals experienced a hypoglycemic-hyperinsulinemic clamp in the morning (AM clamp) and again in the afternoon (PM clamp). BRS was assessed using the modified Oxford method (sequential administration of nitroprusside and phenylephrine) before the initiation of each hypoglycemic-hyperinsulinemic clamp, during the last 30 minutes of hypoglycemia, and the following day. Results: The study included 16 individuals with a diagnosis of T2DM (6 men and 10 women) with the following characteristics (mean ± SD): age 45 ± 11 years, BMI 32 ± 6 kg/m2, hemoglobin A1c 6.4 ± 0.9%, and diabetes duration 8 ± 5 years. At baseline, individuals with T2DM had a BRS of 7.7 ± 3.6 ms/mmHg, which is reduced compared to a BRS of 21.3 ± 13.8 ms/mmHg in a historical healthy control group of 54 subjects (p<0.001). A mixed effects model adjusting for sex, age, BMI, and insulin level, demonstrated a significant effect of hypoglycemia on BRS (p=0.008). Pairwise comparisons revealed a significant decrease in BRS during the PM clamp as compared with BRS assessed at baseline (5.5 ± 5.0 vs 7.7 ± 3.6 ms/mmHg, p=0.045), during the AM clamp (5.5 ± 5.0 vs 10.5 ± 5.6 ms/mmHg, p=0.43), and the following day (5.5 ± 5.0 vs 8.6 ± 3.8 ms/mmHg, p=0.33). Of note, there was no significant difference between BRS at baseline and during the AM clamp (p=0.44). Additionally, insulin levels positively and strongly correlated with BRS at baseline (R2 0.52) and during the AM clamp (R2 0.67). Discussion: Insulin may have a stimulatory effect on BRS during a hyperinsulinemic hypoglycemic clamp that counters the inhibitory effect of hypoglycemia during the first episode of hypoglycemia. However, with exposure to a second hypoglycemic clamp, BRS decreases. Conclusion: Individuals with T2DM have low BRS at baseline, which is further reduced after 2 episodes of hypoglycemia.

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.

The Effect of Hypoglycemia on Spectral Moments in EEG Epochs of Different Durations in Type 1 Diabetes Patients.

The potential of using an electroencephalogram (EEG) to detect hypoglycemia in patients with type 1 diabetes (T1D) has been investigated in both time and frequency domains. Under hyperinsulinemic hypoglycemic clamp conditions, we have shown that the brain's response to hypoglycemic episodes could be described by the centroid frequency and spectral gyration radius evaluated from spectral moments of EEG signals. The aim of this paper is to investigate the effect of hypoglycemia on spectral moments in EEG epochs of different durations and to propose the optimal time window for hypoglycemia detection without using clamp protocols. The incidence of hypoglycemic episodes at night time in five T1D adolescents was analyzed from selected data of ten days of observations in this study. We found that hypoglycemia is associated with significant changes (P < 0.05) in spectral moments of EEG segments in different lengths. Specifically, the changes were more pronounced on the occipital lobe. We used effect size as a measure to determine the best EEG epoch duration for the detection of hypoglycemic episodes. Using Bayesian neural networks, this study showed that 30 second segments provide the best detection rate of hypoglycemia. In addition, Clarke's error grid analysis confirms the correlation between hypoglycemia and EEG spectral moments of this optimal time window, with 86% of clinically acceptable estimated blood glucose values. These results confirm the potential of using EEG spectral moments to detect the occurrence of hypoglycemia.

Ghrelin Protects Against Insulin-Induced Hypoglycemia in a Mouse Model of Type 1 Diabetes Mellitus

Insulin-induced hypoglycemia is a major limiting factor in maintaining optimal blood glucose in patients with type 1 diabetes and advanced type 2 diabetes. Luckily, a counterregulatory response (1) system exists to help minimize and reverse hypoglycemia, although more studies are needed to better characterize its components. Recently, we showed that the hormone ghrelin is permissive for the normal CRR to insulin-induced hypoglycemia when assessed in mice without diabetes. Here, we tested the hypothesis that ghrelin also is protective against insulin-induced hypoglycemia in the streptozotocin (2) mouse model of type 1 diabetes. STZ-treated ghrelin-knockout (KO) (3) mice as well as STZ-treated wild-type (WT) littermates were subjected to a low-dose hyperinsulinemic-hypoglycemic clamp procedure. The STZ-treated ghrelin-KO mice required a much higher glucose infusion rate than the STZ-treated WT mice. Also, the STZ-treated ghrelin-KO mice exhibited attenuated plasma epinephrine and norepinephrine responses to the insulin-induced hypoglycemia. Taken together, our data suggest that without ghrelin, STZ-treated mice modeling type 1 diabetes are unable to mount the usual CRR to insulin-induced hypoglycemia.

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.

High-intensity training as a novel treatment for impaired awareness of hypoglycaemia in type 1 diabetes [HIT4HYPOS]: Protocol for a randomized parallel-group study.

AimThis pilot study aimed to investigate whether a 4‐week programme of intermittent high‐intensity training (HIT) will improve counterregulatory responses and improve hypoglycaemia awareness in adults with type 1 diabetes who have been exposed to recurrent hypoglycaemia.MethodsAdults with type 1 diabetes who have been exposed to recurrent hypoglycaemia will be recruited from NHS Tayside, Scotland. All participants have a 4‐week run‐in period to optimize glycaemic control and to receive instruction in hypoglycaemia avoidance using insulin dose adjustment and real‐time continuous glucose monitoring (CGM). Following this, they will undergo a baseline 90‐minute hyperinsulinaemic hypoglycaemic clamp to assess symptomatic, cognitive and hormonal counterregulatory responses. Subsequently, participants will be randomized in a parallel‐group design to either undergo a 4‐week intervention with HIT or to no exercise with both groups using CGM throughout and receiving additional advice on hypoglycaemia avoidance. Participants in the HIT arm of the trial will be instructed to exercise 3 times a week on a cycle ergometer and asked to achieve ≥ 90% max heart rate during each period of exercise. On completion of the intervention period, all subjects then undergo a second matched hyperinsulinaemic hypoglycaemic clamp study.DiscussionThis pilot study will determine whether high‐intensity exercise may offer a novel approach to restore hypoglycaemic awareness in type 1 diabetes (International Standard Randomised Controlled Trials No: ISRCTN15373978).

Lixisenatide in type 1 diabetes: A randomised control trial of the effect of lixisenatide on post-meal glucose excursions and glucagon in type 1 diabetes patients.

AimsThe GLP1 agonist lixisenatide is glucagonostatic and reduces post‐prandial blood glucose (PPBG) in type 2 diabetes. This study investigates its impact in type 1 diabetes (T1D).MethodsIn a blinded, crossover trial, 25 patients with T1D were randomised to 4 weeks adjunctive treatment with lixisenatide (L) or placebo (P), with a 4‐week washout period. The primary outcome was percentage of 3 hours PPBG in target (4‐10 mmol/L) assessed by CGM before and after treatment. Participants also underwent post‐treatment standardised mixed meal test (MMT, n = 25) and hyperinsulinaemic hypoglycaemic clamp (n = 15).ResultsPPBG CGM readings in target were similar between L vs P (Mean % ± SE, breakfast 45.4 ± 6.0 vs 44.3 ± 6.0, P = .48, lunch 45.5 ± 5.8 vs 50.6 ± 5.3, P = .27 and dinner 43.0 ± 6.7 vs 47.7 ± 5.6, P = .30). HbA1C was similar between L vs P (64.7 ± 1.6 vs 64.1 ± 1.6 mmol/mol, P = .30). Prandial insulin fell after lixisenatide (dose change −0.7 ± 0.6 vs +2.4 ± 0.7 units/d, P = .004), but basal insulin dose was similar between groups. The post‐MMT glucose area under the curve (AUC) was lower with L than P (392.0 ± 167.7 vs 628.1 ± 132.5 mmol/L × min, P < .001), as was the corresponding glucagon AUC (140.0 ± 110.0 vs 304.2 ± 148.2 nmol/L × min, P < .001). Glucagon and counter‐regulatory hormone values at a blood glucose of 2.4 mmol/L during the hypoglycaemic clamp were similar between L and P.ConclusionIn T1D, PPBG values were not altered by adjunctive lixisenatide although prandial insulin dose fell. Glucose and glucagon level during an MMT were significantly lower after lixisenatide, without affecting counter‐regulatory response during hypoglycaemia.

1892-P: Meal- and Glucose-Induced Suppression of Ghrelin Release Is Mediated Primarily by Ghrelin Cell-Expressed Insulin Receptors

Objective: The hormone ghrelin both regulates and is regulated by food intake and blood glucose. Yet, the mechanisms restricting ghrelin release after meals or upon glucose delivery have remained elusive. Here, we tested whether meal and glucose-associated rises in plasma insulin act via ghrelin cell-expressed insulin receptors to suppress ghrelin release. Methods: We generated ghrelin cell-selective insulin receptor-knock out (GIRKO) mice by crossing ghrelin-Cre mice with floxed-insulin receptor mice. We examined ghrelin release using hyperinsulinemic-hypoglycemic clamps, glucose tolerance tests (GTTs), a fast-refeed protocol, and gastric mucosal cell primary cultures. Results: During clamps, plasma ghrelin fell in WT control groups (by 85%) but not in GIRKO mice, leading to a 29% lower glucose infusion rate in GIRKO mice. During GTTs, plasma ghrelin was suppressed in controls (by 42%) but not in GIRKO mice. During the refeed phase of a fast-refeed protocol, plasma ghrelin fell in controls (by 49%) but not in GIRKO mice. Also, insulin suppressed ghrelin release in isolated ghrelin cells of controls (by 19%) but not of GIRKO mice. Conclusions: These data suggest that insulin, acting via ghrelin cell-expressed insulin receptors, is a key mediator of ghrelin release in response to meals, glucose administration, and insulin infusion. Further, suppressed ghrelin release resulting from direct insulin action on ghrelin cells restricts ghrelin’s capacity to protect against hypoglycemia during over-insulinization. Disclosure K. Shankar: None. S. Takemi: None. D. Gupta: None. B.K. Mani: None. S. Osborne-Lawrence: None. N. Metzger: None. E. Berglund: None. J.M. Zigman: Stock/Shareholder; Self; Medtronic. Funding National Institutes of Health (R01DK119341-01A1, R01DK103884 to J.M.Z.), (R01DK109408 to E.B.), (R01DK119341 to J.M.Z., E.B.); David and Teresa Disiere Foundation (to J.M.Z.); Diana and Richard C. Strauss Professorship in Biomedical Research; Mr. and Mrs. Bruce G. Brookshire Professorship in Medicine; Kent and Jodi Foster Distinguished Chair in Endocrinology (to J.M.Z.); Uehara Memorial Foundation (to S.T.)

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)

159-OR: Vitamin E Treatment Reduces Severe Hypoglycemia-Induced Fatal Cardiac Arrhythmias in Type 1 Diabetic Rats

Severe hypoglycemia can lead to fatal cardiac arrhythmias. Our studies have shown that diabetes, per se, increases the risk of hypoglycemia-induced mortality in rats. It was hypothesized that excess oxidative stress, associated with diabetes, increases the heart’s susceptibility to hypoglycemia-induced fatal arrhythmias. To test this hypothesis, Sprague Dawley rats were made diabetic (streptozotocin 65 mg/kg) and randomized to two treatment groups: 1) antioxidant Vitamin E (400 mg/kg/day; n=18) or 2) control (vehicle, n=16) injected subcutaneously over 8 days. Then, rats underwent hyperinsulinemic (0.4 units/kg/min) severe hypoglycemic (10-15 mg/dl) clamps for 3 hours with continuous electrocardiogram recording. Confirming its antioxidant properties, Vitamin E treatment significantly reduced oxidative stress in the heart 3.3-fold versus controls (Figure). As compared to hypoglycemia-induced mortality of 38% in controls, Vitamin E treatment significantly reduced mortality to 6% (p&amp;lt;0.05; Figure). Additionally, Vitamin E treatment reduced 3rd degree heart block (6%) vs. control (46%; Figure). Overall, these results suggest that 1) oxidative stress in diabetes increases the heart’s susceptibility to hypoglycemia-induced arrhythmias, and 2) Vitamin E treatment reduces oxidative stress and reduces both cardiac arrhythmias and mortality to insulin-induced severe hypoglycemia. Disclosure C.M. Reno: None. M.B. Oxspring: None. J. Bayles: None. I. Holiday: None. Y. Huang: None. S.J. Fisher: None. Funding National Institutes of Health (5T32DK091317, R01NS070235); JDRF (3-APF-2017-407-A-N)

1774-P: Neurally Mediated Prandial Islet-Cell Function Is Glucose-Independent and Preserved after Gastric Bypass and Sleeve Gastrectomy

The weight-loss independent glycemic effects of gastric bypass (GB) are characterized by prandial short-lived hyperinsulinemia as a result of β-cell stimulation by glucose and incretins. To investigate the effect of parasympathetic nervous system (PNS) activity, a neural component of the enteroinsular axis, on islet function, 9 subjects with history of GB, 6 with prior sleeve gastrectomy (SG), and 4 non-surgical controls were recruited. The GB, SG, and CN groups were matched for BMI, FFM, A1C, and age; and the GB and SG for weight loss and time postsurgery; none had diabetes. To measure islet hormone responses to meal ingestion when the effect of GI hormones and glycemia is minimal, subjects consumed a liquid meal (280 kcal, 20% glucose) at 2 hr of a 5-hr hyperinsulinemic (120 mU/m2/min) hypoglycemic (55mg/dl) clamp with and without atropine infusion on two separate studies. Fasting levels of glucose, islet and gut hormones were similar among 3 groups and 2 studies. Despite similar insulin sensitivity among the groups the insulin clearance estimated before meal ingestion was larger in surgical patients (P=0.08). Suppression of endogenous β-cell secretion by hypoglycemia was less in both surgical groups compared to controls (P&amp;lt;0.001), but it was reduced by atropine in 3 groups (P&amp;lt;0.05). Hypoglycemia-induced glucagon secretion was not affected by PNS blockade. Blocking PNS diminished both β- and α-cell secretory response to meal ingestion (P &amp;lt; 0.05) in both surgical and non-surgical groups. Pre- and post-meal pancreatic polypeptide, marker of vagal input to the islet, declined to baseline values during atropine infusion. Meal-induced GIP secretion was similar in studies with and without atropine whereas blocking PNS significantly reduced GLP-1 secretion (P &amp;lt; 0.05) in all subjects. Our data shows that in nondiabetic subjects with prior GB and SG, PNS activity regulates islet and gut hormone secretory response to meal ingestion independent of glucose levels. Disclosure H. Honka: None. A.M. Al Zubaidi: None. R.A. DeFronzo: None. A. Gastaldelli: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Gilead Sciences, Inc., Inventiva Pharma, Novo Nordisk Inc. M. Salehi: None. Funding Finnish Cultural Foundation (00180071); National Institutes of Health (DK105379)