Hypoglycemic Counterregulation Research Articles

Glutamate Prevents Altered Mitochondrial Function Following Recurrent Low Glucose in Hypothalamic but Not Cortical Primary Rat Astrocytes.

Astrocytes contribute to glutamatergic signalling, which is required for hypoglycaemia counterregulation and is impaired by recurrent insulin-induced hypoglycaemia. This study examined the glutamate response of astrocytes when challenged with acute and recurrent low glucose (RLG) exposure. The metabolic responses of cortical (CRTAS) and hypothalamic (HTAS) primary rat astrocytes were measured in acute and recurrent low glucose using extracellular flux analyses. RLG caused mitochondrial adaptations in both HTAS and CRTAS, many of which were attenuated by glutamate exposure during low glucose (LG) treatments. We observed an increase in capacity of HTAS to metabolise glutamine after RLG exposure. Demonstrating astrocytic heterogeneity in the response to LG, CRTAS increased cellular acidification, a marker of glycolysis in LG, whereas this decreased in HTAS. The directional change in intracellular Ca2+ levels of each cell type, correlated with the change in extracellular acidification rate (ECAR) during LG. Further examination of glutamate-induced Ca2+ responses in low glucose treated CRTAS and HTAS identified sub-populations of glucose-excited- and glucose-inhibited-like cells with differing responses to glutamate. Lastly, release of the gliotransmitter ATP by HTAS was elevated by RLG, both with and without concurrent glutamate exposure. Therefore, hypothalamic astrocytes adapt to RLG by increasing glutamate uptake and oxidation in a manner that prevents RLG-induced mitochondrial adaptations.

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.

Measures of glucose homeostasis during and after duodenal exclusion using a duodenal-jejunal bypass liner in a normoglycemic, nonobese canine model.

Discovering the role duodenal exclusion plays in weight loss and resolution of type 2 diabetes (T2D) may help refine the surgical and nonsurgical treatment of obesity and T2D. To assess changes in glucose homeostasis due to duodenal exclusion using a duodenal-jejunal bypass liner (DJBL) in a nonobese canine model. Academic laboratory setting. An intravenous glucose tolerance test (IVGTT), and a mixed-meal tolerance test (MMTT) at baseline, 1, and 6 weeks post DJBL implantation (I1 and I6, respectively), and 1 and 6 weeks post DJBL removal (R1 and R6, respectively) were done in canines (n = 7) fed a normal chow diet. Placement of the DJBL induced weight loss that was maintained until 4 weeks post removal (R4), despite normal food intake. Total bile acids (TBA) and glucagon-like peptide-1 (GLP-1) during the MMTT were significantly increased at I1 and were associated with increased lactate and free fatty acids. Hypoglycemia counter-regulation was blunted during the IVGTT at I1 and I6, returning to baseline at R1. While there were no changes to insulin sensitivity during the experiment, glucose tolerance was significantly increased following the removal of the DJBL at R1. These data show that in a normoglycemic, nonobese canine model, duodenal exclusion induces energy intake-independent weight loss and negative metabolic effects that are reversed following re-exposure of the small intestine to nutrients.

164-OR: Metoclopramide Restores the Counterregulatory Response to Hypoglycemia in Rodents

To test the hypothesis that metoclopramide (a dopamine antagonist) may restore hypoglycemic counterregulation, 10-week old rats underwent recurrent (insulin-induced) hypoglycemia (RH) to induce a blunted sympathoadrenal response. RH rats were treated with vehicle or metoclopramide (RH+MET: 3 mg/kg IP) . A third control group did not undergo recurrent hypoglycemia (RS: recurrent saline) . MET was administered either during RH phase (concurrent protocol) or after RH (post-induction protocol) . All rats (n=7-14/group) underwent a hyperinsulinemic (50mU/kg/min) hypoglycemic (45±5 mg/dl) clamp. In the concurrent protocol, epinephrine response to hypoglycemia in RS (1434±275 pg/ml) was blunted in RH (460±1pg/ml, p<0.vs. RS) and increased with MET (1072±141 pg/ml, p<0.vs. RH) . In the post-induction protocol, epinephrine in RS (1949±413 pg/ml) was again blunted in RH (581±199 pg/ml, p<0.vs. RS) and increased with MET (1460±202 pg/ml, P<0.vs. RH) . In summary, 1) metoclopramide prevented the development of an impaired sympathoadrenal response to hypoglycemia, and 2) in rodents that had an impaired response to hypoglycemia, subsequent metoclopramide treatment restored the sympathoadrenal response. In conclusion, 1) dopaminergic signaling plays a role in the development of impaired counterregulation, and 2) post hoc treatment with metoclopramide improves the sympathoadrenal response to hypoglycemia. Disclosure M.H.Devore: None. S.Sharma: None. S.Fisher: None. Funding NIH R01DK118082

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)

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.

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.

Leptin receptor signaling is required for intact hypoglycemic counterregulation: A study in male Zucker rats.

Hypoglycemia is a major barrier to clinical management of persons with diabetes. Emerging evidence supports a role for leptin in gating hypoglycemic counterregulation. This work demonstrates that male leptin receptor null, Zucker (fa/fa), rats display severe impairments in hypoglycemic counterregulation. Thus, augmenting leptin levels may have clinical utility for preventing hypoglycemia.

342-P: Effects of Sitagliptin on Hypoglycemic Episodes and Counterregulation in Type 2 Diabetic Patients Tightly Titrated with Insulin Glargine

DPP-4 inhibitors may prevent hypoglycemic episodes in insulin-treated type 2 diabetes (T2D), but mechanisms are unclear. 20 T2D patients treated with basal insulin and metformin (± other agents) were recruited for two randomized treatments (placebo or 100 mg/d sitagliptin) in addition to insulin glargine/metformin; 17 completed (3 women, 14 men; age 57 ± 7 yrs.; BMI 28.8 ± 3.9 kg/m2; HbA1c 7.4 ± 0.7 %). After titrating fasting plasma glucose (PG) to 90-110 mg/dl, patients were observed during a 3-day (d) inpatient period with exposure to oral glucose or lipid loads, strenuous exercise and fasting, while increasing insulin doses to 110, and 120 % of the titrated dose before d 2 and 3, respectively. Hypoglycemic episodes (PG < 70 mg/dl) were identified by measuring venous plasma glucose or symptoms, and prompted the determination of counter-regulatory hormones at the glucose nadir and 30 and 60 min later. After titrating insulin glargine, fasting PG was 112 ± 5 and 107 ± 5 mg/dl with placebo and sitagliptin treatment, respectively. Concentrations of insulin, C-peptide and counterregulatory hormones were similar during both treatments. Hypoglycemic episodes were mainly nocturnal (10 p.m. to 7 a.m.), with a similar PG nadir (65 ± 4 mg/dl) and incidence with placebo and sitagliptin (4.5 ± 3.2 vs. 4.6 ± 3.6 episodes per patient, respectively, p = 0.96). With a similar incidence of daytime (7 a.m. to 10 p.m.) chemical hypoglycemia, the incidence of symptomatic episodes was higher with sitagliptin (0.2 ± 0.4 vs. 0.0 ± 0.0, p = 0.028). Counter-regulatory responses were suppressed by sitagliptin for cortisol (p = 0.022), unchanged for glucagon, HGH, noradrenaline and prolactin, and accentuated for adrenaline (p = 0.022). Our results suggest that sitagliptin treatment augments counter-regulatory responses of adrenaline at minimal degrees of hypoglycemia. This may increase symptoms, in particular during daytime, and may offer opportunities for earlier treatment. Disclosure M. A. Nauck: Advisory Panel; Self; Berlin-Chemie AG, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk, Consultant; Self; Eli Lilly and Company, Novo Nordisk, Research Support; Self; Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk, Speaker’s Bureau; Self; Berlin-Chemie AG, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novo Nordisk. A. Feldmann: None. J. J. Meier: Advisory Panel; Self; AstraZeneca, MSD Corporation, Novo Nordisk, Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, MSD Corporation, Novo Nordisk, Sanofi. M. Nauck: None. C. Kapitza: Research Support; Self; ADOCIA, Afon Technology, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestlé, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Funding Merck Sharp & Dohme Corp

Liver glycogen-induced enhancements in hypoglycemic counterregulation require neuroglucopenia.

Iatrogenic hypoglycemia is a prominent barrier to achieving optimal glycemic control in patients with diabetes, in part due to dampened counterregulatory hormone responses. It has been demonstrated that elevated liver glycogen content can enhance these hormonal responses through signaling to the brain via afferent nerves, but the role that hypoglycemia in the brain plays in this liver glycogen effect remains unclear. During the first 4 h of each study, the liver glycogen content of dogs was increased by using an intraportal infusion of fructose to stimulate hepatic glucose uptake (HG; n = 13), or glycogen was maintained near fasting levels with a saline infusion (NG; n = 6). After a 2-h control period, during which the fructose/saline infusion was discontinued, insulin was infused intravenously for an additional 2 h to bring about systemic hypoglycemia in all animals, whereas brain euglycemia was maintained in a subset of the HG group by infusing glucose bilaterally into the carotid and vertebral arteries (HG-HeadEu; n = 7). Liver glycogen content was markedly elevated in the two HG groups (43 ± 4, 73 ± 3, and 75 ± 7 mg/g in NG, HG, and HG-HeadEu, respectively). During the hypoglycemic period, arterial plasma glucose levels were indistinguishable between groups (53 ± 2, 52 ± 1, and 51 ± 1 mg/dL, respectively), but jugular vein glucose levels were kept euglycemic (88 ± 5 mg/dL) only in the HG-HeadEu group. Glucagon and epinephrine responses to hypoglycemia were higher in HG compared with NG, whereas despite the increase in liver glycogen, neither increased above basal in HG-HeadEu. These data demonstrate that the enhanced counterregulatory hormone secretion that accompanies increased liver glycogen content requires hypoglycemia in the brain.NEW & NOTEWORTHY It is well known that iatrogenic hypoglycemia is a barrier to optimal glycemic regulation in patients with diabetes. Our data confirm that increasing liver glycogen content 75% above fasting levels enhances hormonal responses to insulin-induced hypoglycemia and demonstrate that this enhanced hormonal response does not occur in the absence of hypoglycemia in the brain. These data demonstrate that information from the liver regarding glycogen availability is integrated in the brain to optimize the counterregulatory response.

Acceleration of Gastric Emptying by Insulin-Induced Hypoglycemia is Dependent on the Degree of Hypoglycemia.

Hypoglycemia is a major barrier to optimal glycemic control in insulin-treated diabetes. Recent guidelines from the American Diabetes Association have subcategorized "non-severe" hypoglycemia into level 1 (<3.9 mmol/L) and 2 (<3 mmol/L) hypoglycemia. Gastric emptying of carbohydrate is a major determinant of postprandial glycemia but its role in hypoglycemia counter-regulation remains underappreciated. "Marked" hypoglycemia (~2.6 mmol/L) accelerates gastric emptying and increases carbohydrate absorption in health and type 1 diabetes, but the impact of "mild" hypoglycemia (3.0-3.9 mmol/L) is unknown. To determine the effects of 2 levels of hypoglycemia, 2.6 mmol/L ("marked") and 3.6 mmol/L ("mild"), on gastric emptying in health. Fourteen healthy male participants (mean age: 32.9 ± 8.3 years; body mass index: 24.5 ± 3.4 kg/m2) from the general community underwent measurement of gastric emptying of a radiolabeled solid meal (100 g beef) by scintigraphy over 120 minutes on 3 separate occasions, while blood glucose was maintained at either ~2.6 mmol/L, ~3.6 mmol/L, or ~6 mmol/L in random order from 15 minutes before until 60 minutes after meal ingestion using glucose-insulin clamp. Blood glucose was then maintained at 6 mmol/L from 60 to 120 minutes on all days. Gastric emptying was accelerated during both mild (P = 0.011) and marked (P = 0.001) hypoglycemia when compared to euglycemia, and was more rapid during marked compared with mild hypoglycemia (P = 0.008). Hypoglycemia-induced gastric emptying acceleration during mild (r = 0.57, P = 0.030) and marked (r = 0.76, P = 0.0014) hypoglycemia was related to gastric emptying during euglycemia. In health, acceleration of gastric emptying by insulin-induced hypoglycemia is dependent on the degree of hypoglycemia and baseline rate of emptying.

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)

4447 Leptin supplementation prevents the loss of hypoglycemia-induced glucagon release following exposure to six days of severe caloric restriction in mice

OBJECTIVES/GOALS: We have recently shown that mice exposed to six days of 60% caloric restriction acutely display reduced hypoglycemia-induced glucagon release following refeeding, and that this effect is concurrent with low leptin levels. The current study was conducted to ascertain if leptin treatment during caloric restriction would reverse this effect. METHODS/STUDY POPULATION: Three groups of mice were used, an ad libitum (Ad-lib) fed group and two caloric restriction (CR) groups, one of which received twice daily leptin injection (0.5-1μg/g; IP) and the other vehicle (saline) during their caloric restriction. CR mice were placed on 60% caloric restriction for 6 consecutive days. Ad lib mice were housed in an identical manner but fed ad libitum during this same period. Following 6 days of restriction, CR mice were given ad lib access to food for 16 h. After the 16 h period of refeeding, both CR and ad lib mice began a 6 h fast which was immediately followed by a hypoglycemic insulin tolerance test (ITT). ITTs consisted of a variable dose of insulin intended to achieve a blood glucose of ~45 mg/dL within 60 minutes, at which time blood was collected for glucagon and corticosterone assays. RESULTS/ANTICIPATED RESULTS: The mean blood glucose levels during the ITT at 45 and 60 minutes post injection across all three groups were 46.8 + 3.1 and 37.0 + 2.4, respectively. There were no significant differences in glucose levels between the three groups at these two time points. As expected, saline treated CR mice displayed significantly reduced serum glucagon levels in response to the ITT relative to Ad-lib mice (23.5 + 10.9 vs. 91.7 + 20.8 pg/mL, p = 0.009). In contrast, leptin-treated CR mice maintained their hypoglycemia-induced glucagon response to the ITT (78.0 + 16.8 pg/mL, p&gt;0.99 vs. Ad-lib group). In addition, although corticosterone levels in saline treated CR mice were numerically lower than in Ad-lib mice, this difference was not statistically significance (3928 + 277 vs. 4571 + 178 pg/mL, p = 0.179). DISCUSSION/SIGNIFICANCE OF IMPACT: Diabetes patients on insulin therapy often develop impaired hypoglycemic counter-regulation which can lead to life-threatening hypoglycemic complications. Our results suggest that leptin may hold promise as a therapeutic intervention for the prevention of impaired hypoglycemic counter-regulation in persons with diabetes.

158-OR: Calcium Channel Blockade Protects against Severe Hypoglycemia–Induced Fatal Cardiac Arrhythmias in Rats

It was hypothesized that in response to insulin-induced severe hypoglycemia, excess calcium influx into the heart causes fatal cardiac arrhythmias. To test if blocking the calcium channels would decrease fatal cardiac arrhythmias during severe hypoglycemia, L-type calcium channel blocker (Verapamil; 1 mg/kg, n = 25) or saline (n = 24) were infused into Sprague Dawley rats during a hyperinsulinemic (0.2 mU/kg/min) severe hypoglycemic (10-15 mg/dl) clamp for 3 hours with ECG. During severe hypoglycemia, verapamil completely prevented mortality compared to 21% mortality in controls (p &amp;lt; 0.05; Figure). Decreased mortality was associated with a 99% decrease in 2nd degree heart block (Figure) and prevention of 3rd degree heart block compared to saline (p &amp;lt; 0.05). Glucagon and epinephrine were similar between the groups suggesting verapamil does not affect hypoglycemic counterregulation. Consistent with the notion of calcium-mediated arrhythmias, separate experiments demonstrated that pharmacological blockade of ryanodine receptor-mediated calcium signaling also reduced heart block by 97% and prevented mortality due to hypoglycemia. In summary, blocking calcium channels protects against severe hypoglycemia-induced fatal cardiac arrhythmias. Blockade of cardiac calcium channels could be a potential approach to prevent arrhythmias in people with diabetes at risk for hypoglycemia. Disclosure C.M. Reno: None. Y. Huang: None. C.G. Christensen: None. M.B. Oxspring: None. J. Bayles: None. S.J. Fisher: None. Funding National Institutes of Health (5T32DK091317, R01NS070235); JDRF (3-APF-2017-407-A-N)

Consuming a ketogenic diet leads to altered hypoglycemic counter-regulation in mice

Ketogenic diets (KDs) are becoming increasingly popular for the treatment of diabetes, yet they are associated with increased frequency of hypoglycemia. Here we report that mice fed a KD display blunted glucagon release to hypoglycemia and neuroglucopenia, suggesting that consuming a KD may increase the risk for iatrogenic hypoglycemia.

Mice with nucleus tractus solitarius injury induced by chronic restraint stress present impaired ability to raise blood glucose and glucagon levels when blood glucose levels plummet.

Chronic restraint stress (CRS) induces insulin-resistant hyperglycemia by inducing injury to the brain neurons in the nucleus tractus solitarius (NTS). However, the CRS mice did not suffer from hypoglycemia. In this study, mice of both CRS and NTS mechanical injury models were induced to investigate whether impaired glucose metabolism has changed upon the extension of the survival time after modeling. Body weight, food and water intake, fasting blood glucose, glucose tolerance, and glucose metabolism related to blood hormone levels were monitored for 12 weeks following the induction of injury. The mice were also administered with insulin intraperitoneally, and the blood glucose and glucagon levels were measured and compared to those in the control mice administered with saline. The results showed that the body weights of CRS-hyperglycemic mice were significantly higher than those in the control group, while the body weights of NTS mechanically injured mice were significantly lower than those in the control group. The food and water intake of both CRS-hyperglycemic and NTS mechanically injured mice were significantly more than those in the control groups. Although the levels of fasting blood glucose and resting serum hormone in the injured mice have returned to normal levels, the utilization of glucose and hypoglycemic counterregulation (the response that raises the blood glucose levels) was impaired in either CRS-hyperglycemic or NTS mechanically injured mice. The blood glucagon levels following insulin administration showed abnormal increase. These findings suggest that the CRS-induced NTS injury resulted not only in early insulin-resistant hyperglycemia but also impaired the ability to raise blood glucose and glucagon levels when blood glucose levels plummet in the later stage.

Evaluating Glial Acetate Metabolism as a Biomarker of Hypoglycemic Counterregulation

Evaluating Glial Acetate Metabolism as a Biomarker of Hypoglycemic Counterregulation

386-P: Chronic and Acute Consumption of a Ketogenic Diet Leads to Deficits in Hypoglycemic Counterregulation in Mice

Background: Due to their metabolic benefits, ketogenic diets (KDs) are growing increasingly popular as a dietary strategy for augmenting treatment of type 1 and type 2 diabetes. Although much is known regarding the metabolic benefits of KDs, little is known about their effect on hypoglycemic counter-regulation. We tested whether mice fed a KD have normal hypoglycemic counter-regulation. Methods: B6 mice were fed a KD (Bio-Serv; F3666) ad lib for either 28 days (chronic) or 7 days (acute). On the final day of the diet, hypoglycemic counter-regulation was assessed via glucoprivic challenge (ICV 2DG, 1 mg/mouse, or saline) in the chronic KD group or via a hypoglycemic insulin tolerance test (ITT) in the acute KD group. Deficits in the counter-regulatory response were determined by comparing glucose and glucagon levels to control mice simultaneously fed a standard chow diet (LabDiet; 5001). ITTs consisted of a variable dose of IP insulin which produced equivalent hypoglycemia in all groups (∼45 mg/dL). Results: As expected, mice fed a KD either acutely or chronically had lower fasting blood glucose levels relative to controls (chronic KD: 132.4 and 167.5mg/dL respectively, p = 0.0011, n= 12-17/group; Acute KD: 167.3 and 200.7 mg/dL respectively, p = 0.026; n = 7-10/group). In response to glucoprivic challenge, chronic KD mice had a blunted rise in both glucose and glucagon relative to chow fed mice (Delta glucose: 84.2 and 135.8 mg/dL respectively, p = 0.017; Glucagon: 32.7 and 73.8 pg/mL respectively, p = 0.027; n = 5-8/group). Acute KD mice also had reduced serum glucagon levels in response to the ITT (28.8 and 50.1 pg/mL respectively, p = 0.044). Conclusions: We show that consuming a KD reduces glucose counter-regulation in mice. If a similar effect exists in persons with diabetes it could blunt a homeostatic response which is critical for counteracting treatment-induced hypoglycemic crises. Further characterization of the effects of KDs on glucose counter-regulation is warranted. Disclosure M.A. DuVall: None. C. Coulter: None. C.M. Hill: None. C. Morrison: None. H.X. Sikaffy: None. D. McDougal: None. Funding American Diabetes Association (1-15-JF-37 to D.M.)

401-P: Acute Low Glucose Alters Human Primary Astrocyte Expression of Endoplasmic Reticulum Stress and Mitochondrial Associated Genes, Which Is Blunted after Recurrent Low Glucose

Astrocytes are important regulatory cells in the brain that support neuronal function. However, their role in hypoglycemia counterregulation is poorly understood. Moreover, astrocytic transcriptional responses to hypoglycemia and the mechanisms by which recurrent hypoglycemia (RH) suppresses counterregulation are not understood. Using human primary astrocytes (HPA) in vitro we tested whether recurrent low glucose (RLG), to model RH, would regulate differential gene expression (DGE). HPA cells were exposed to 3-hour bouts of 2.5 or 0.1 mM glucose over 4 days and mRNA expression was quantified using RNA-sequencing and DGE measured using DESeq2. Acute low glucose (LG) differentially expressed 31 genes including the downregulation of mitochondrial genes (mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 2, subunit 4 and subunit 4L; MT-ND2, MT-ND4 and MT-ND4L) and endoplasmic reticulum stress associated genes (X-box binding protein 1; XBP1, and heat shock protein family A; Hsp70, member 1A, and member 5; HSPA1A and HSPA5). However, neither antecedent RLG (aRLG) nor RLG treated cells have significantly altered gene expression compared to control. LG treatment decreased the expression of XBP1 and increased thioredoxin interacting protein (TXNIP) and exostosin Like glycosyltransferase 3 (EXTL3) which were also similarly altered by low glucose compared to aRLG.MT-ND2, MT-ND4 and MT-ND4L genes are associated with diabetes and neurodegeneration. Furthermore, LG-induced reduction of XBP1 may also increase fatty acid oxidation by disinhibiting inositol-requiring kinase 1α. This corroborates previous work indicating low glucose increases fatty acid utilisation in astrocytes and the hypothalamus. While the functional impact of differential gene expression needs assessing, this provides evidence that these genes may be important in astrocytic responses to hypoglycemia. Disclosure P.G. Weightman Potter: None. S.J. Washer: None. A. Jeffries: None. E.L. Dempster: None. C. Beall: None. Funding Novo Nordisk UK Research Foundation; Diabetes UK; Mary Kinross Charitable Trust

Repetitive hypoglycemia reduces activation of glucose-responsive neurons in C1 and C3 medullary brain regions to subsequent hypoglycemia.

The impaired ability of the autonomic nervous system to respond to hypoglycemia is termed “hypoglycemia-associated autonomic failure” (HAAF). This life-threatening phenomenon results from at least two recent episodes of hypoglycemia, but the pathology underpinning HAAF remains largely unknown. Although naloxone appears to improve hypoglycemia counterregulation under controlled conditions, hypoglycemia prevention remains the current mainstay therapy for HAAF. Epinephrine-synthesizing neurons in the rostroventrolateral (C1) and dorsomedial (C3) medulla project to the subset of sympathetic preganglionic neurons that regulate peripheral epinephrine release. Here we determined whether or not C1 and C3 neuronal activation is impaired in HAAF and whether or not 1 wk of hypoglycemia prevention or treatment with naloxone could restore C1 and C3 neuronal activation and improve HAAF. Twenty male Sprague-Dawley rats (250–300 g) were used. Plasma epinephrine levels were significantly increased after a single episode of hypoglycemia (n = 4; 5,438 ± 783 pg/ml vs. control 193 ± 27 pg/ml, P < 0.05). Repeated hypoglycemia significantly reduced the plasma epinephrine response to subsequent hypoglycemia (n = 4; 2,179 ± 220 pg/ml vs. 5,438 ± 783 pg/ml, P < 0.05). Activation of medullary C1 (n = 4; 50 ± 5% vs. control 3 ± 1%, P < 0.05) and C3 (n = 4; 45 ± 5% vs. control 4 ± 1%, P < 0.05) neurons was significantly increased after a single episode of hypoglycemia. Activation of C1 (n = 4; 12 ± 3%, P < 0.05) and C3 (n = 4; 19 ± 5%, P < 0.05) neurons was significantly reduced in the HAAF groups. Hypoglycemia prevention or treatment with naloxone did not restore the plasma epinephrine response or C1 and C3 neuronal activation. Thus repeated hypoglycemia reduced the activation of C1 and C3 neurons mediating adrenal medullary responses to subsequent bouts of hypoglycemia.