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In people with type 1 diabetes, hypoglycemia can induce cardiac arrhythmias. In rodent experiments, severe hypoglycemia can induce fatal cardiac arrhythmias, especially so in diabetic models. Increased oxidative stress associated with insulin-deficient diabetes was hypothesized to increase susceptibility to severe hypoglycemia-induced fatal cardiac arrhythmias. To test this hypothesis, Sprague-Dawley rats were made insulin deficient with streptozotocin and randomized into two groups

BACKGROUND: This study aimed to understand the prevalence of prediabetes and diabetes in patients with cancer overall, by tumor site, by cancer treatment, and by time point in the cancer continuum. METHODS: This cohort study has been conducted at the Huntsman Cancer Institute, Salt Lake City, Utah. Patients with a first primary invasive cancer enrolled in the Total Cancer Care protocol between July 2016 and July 2018 were eligible for this analysis. The prevalence of prediabetes and diabetes is based on ICD code, laboratory tests for HbA1c (%), fasting plasma glucose (mg/dl), non-fasting blood glucose (mg/dl), or insulin prescription. RESULTS: The final cohort comprised 3,512 patients with cancer, with a mean age of 57.8 years at cancer diagnosis. Of all patients, 49.1% (n=1,724) were female. At cancer diagnosis, the prevalence of prediabetes was 6.0% (95% CI: 5.3–6.8) and of diabetes 12.2% (95% CI: 11.2–13.3). One year after diagnosis the prevalences were 16.6% for prediabetes (95% CI: 15.4–17.9) and 25.0% for diabetes (95% CI: 23.6–26.4). At the end of the observation period, the prevalence of prediabetes was 21.2% (95% CI: 20.4–23.0) and of diabetes was 32.6% (95% CI: 29.2–32.1). Patients with myeloma (39.2%; 95% CI: 32.6–46.2) had the highest prevalence of prediabetes and patients with pancreatic cancer the highest prevalence of diabetes (65.1%; 95% CI: 57.0–72.3). In patients who underwent chemotherapy (prediabetes: 29.1% vs 15.6%; diabetes: 37.6% vs 29.0%), radiation therapy (prediabetes: 24.3% vs. 19.9%; diabetes: 33.7% vs 32.1%), or immunotherapy (diabetes: 29.2% vs 20.4; prediabetes: 36.0% vs 32.2%), the prevalence of prediabetes and diabetes was higher compared to patients not undergoing those therapies. CONCLUSIONS: Every second patient with cancer suffers from prediabetes or diabetes. It is essential to foster interprofessional collaboration and to develop evidence-based practice guidelines. A better understanding of the impact of cancer treatment on the development of prediabetes and diabetes remains critical.

Sudden death related to hypoglycemia is thought to be due to cardiac arrhythmias. A clearer understanding of the cardiac changes associated with hypoglycemia is needed to reduce mortality. The objective of this work was to identify distinct patterns of electrocardiogram heartbeat changes that correlated with glycemic level, diabetes status, and mortality using a rodent model. Electrocardiogram and glucose measurements were collected from 54 diabetic and 37 non-diabetic rats undergoing insulin-induced hypoglycemic clamps. Shape-based unsupervised clustering was performed to identify distinct clusters of electrocardiogram heartbeats, and clustering performance was assessed using internal evaluation metrics. Clusters were evaluated by experimental conditions of diabetes status, glycemic level, and death status. Overall, shape-based unsupervised clustering identified 10 clusters of ECG heartbeats across multiple internal evaluation metrics. Several clusters demonstrating normal ECG morphology were specific to hypoglycemia conditions (Clusters 3, 5, and 8), non-diabetic rats (Cluster 4), or were generalized among all experimental conditions (Cluster 1). In contrast, clusters demonstrating QT prolongation alone or a combination of QT, PR, and QRS prolongation were specific to severe hypoglycemia experimental conditions and were stratified heartbeats by non-diabetic (Clusters 2 and 6) or diabetic status (Clusters 9 and 10). One cluster demonstrated an arrthymogenic waveform with premature ventricular contractions and was specific to heartbeats from severe hypoglycemia conditions (Cluster 7). Overall, this study provides the first data-driven characterization of ECG heartbeats in a rodent model of diabetes during hypoglycemia.

Background: Continuous glucose monitoring (CGM) systems help reduce hypoglycemia in patients with type 1 diabetes (T1D). It remains unclear whether T1D patients with impaired awareness of hypoglycemia (IAH) continue to develop more hypoglycemia than those with normal hypoglycemia awareness (NA) despite CGM use. Materials and Methods: For this cross-sectional observational study, 99 T1D patients using real-time CGMs for ≥86% of time were recruited. Fifty and 49 patients were found to have NA and IAH (based on the Clarke questionnaire), respectively. Two-week CGM hypoglycemia data were collected. Results: IAH was associated with greater percentages of CGM values

Problem American Indian/Alaska Native (AI/AN) populations are facing multiple health crises, including limited access to care, high rates of chronic disease, and early mortality that is far worse than other underrepresented minorities in the U.S. According to the Association of American Indian Physicians, AI/AN people represent 2.0% of the U.S. population but only 0.2% of medical students and 0.1% of full-time faculty at MD-granting institutions. Increasing the number of AI/AN clinicians and scientists is one strategy to improve health outcomes in the AI/AN population and address these crises. Approach In 2010, the University of Utah partnered with research, cultural, and professional mentors to create a 10-week summer Native American Research Internship (NARI) program for AI/AN college students across the U.S. who are interested in pursuing biomedical careers. NARI attracts and supports AI/AN students by offering mentored summer research internships in an innovative, culturally-aware framework that adapts to observed challenges to optimize educational experiences and support biomedical career aspirations. Outcomes During the first decade of the NARI program, 128 students from 22 U.S. states, representing 46 tribal nations and 57 colleges and universities, have participated. Of those 128 students, 113 (88%) have completed a bachelor's degree and the remaining 15 (12%) are currently working towards a bachelor's degree. No NARI student has dropped out of college. Twenty-six (20%) NARI alumni have matriculated to medical school and 30 (23%) to graduate school. Eight (6%) participants have completed medical school and 3 (2%) participants are pursuing a PhD in science. An additional 36 (28%) have gained employment in biomedical research fields. Next steps The NARI program has increased the participation of AI/AN students in medicine and the biomedical sciences. The innovative, culturally-aware, and adaptive framework is a model for other programs for AI/AN students and students in other underrepresented communities.

OBJECTIVE This study aimed to 1) identify the frequency of severe and level 2 hypoglycemia presenting in individuals with type 1 diabetes using continuous glucose monitoring systems (CGMs), including those with concomitant closed-loop insulin pumps, in a clinical practice setting and 2) evaluate the impact of beliefs around hypoglycemia in the development of severe and level 2 hypoglycemia in this population. RESEARCH DESIGN AND METHODS A cross-sectional survey study in adults with type 1 diabetes using CGMs >6 months was conducted at a large tertiary academic center. Participant demographics, 6-month severe hypoglycemia history, hypoglycemia beliefs (with the Attitude to Awareness of Hypoglycemia questionnaire), and 4-week CGM glucose data were collected. Statistical analysis was performed to assess the presentation of severe and level 2 hypoglycemia and identify associated risk factors. RESULTS A total of 289 participants were recruited (including 257 participants with CGM data within the last 3 months). Of these, 25.6% experienced at least one severe hypoglycemic episode in the last 6 months, and 13.6% presented with ≥1% of time in level 2 hypoglycemia on CGMs. Reporting beliefs about prioritizing hyperglycemia avoidance was associated with severe hypoglycemia development (P < 0.001), while having beliefs of minimal concerns for hypoglycemia was associated with spending ≥1% of time in level 2 hypoglycemia (P = 0.038). CONCLUSIONS Despite the use of advanced diabetes technologies, severe and level 2 hypoglycemia continues to occur in individuals with type 1 diabetes and high hypoglycemia risks. Human factors, including beliefs around hypoglycemia, may continue to impact the effectiveness of glucose self-management.

Targeted suppression of autoimmune diseases without collateral suppression of normal immunity remains an elusive yet clinically important goal. Targeted blockade of the programmed death-1 receptor (PD-1) — an immune checkpoint factor expressed by activated T cells and B cells — is an efficacious therapy for potentiating immune activation against tumours. Here, we show that an immunotoxin consisting of an anti-PD-1 single-chain variable fragment, an albumin-binding domain and Pseudomonas exotoxin targeting PD-1-expressing cells selectively recognizes and induces the killing of the cells. Administration of the immunotoxin to mouse models of autoimmune diabetes delays disease onset, and its administration in mice paralyzed by experimental autoimmune encephalomyelitis ameliorates symptoms. In all mouse models, the immunotoxin reduced the numbers of PD-1-expressing cells, of total T cells and of cells of an autoreactive T cell clone found in inflamed organs, while maintaining active adaptive immunity, as evidenced by full-strength immune responses to vaccinations. The targeted depletion of PD-1-expressing cells contingent to the preservation of adaptive immunity might be effective in the treatment of a wide range of autoimmune diseases.

Due to limitations in access to specialized eye care, the majority of people with diabetic retinopathy (DR) are not screened routinely. To increase screening for DR, the Utah Diabetes and Endocrinology Center clinic began to offer point-of-care retinal image screening for patients with diabetes who had not had a dilated eye exam in the last year. Retinal photos obtained with a digital non-mydriatic camera were interpreted by a vitreoretinal specialist. From 11/2017 to 12/2020, 320 people underwent point-of-care DR screening. Of these, 298 (93%) had retinal images that were of sufficient quality. Mild-moderate DR was identified in 63 participants (21%), moderate-severe DR in 29 participants (10%) and proliferative DR in 11 participants (4%). Patients with advanced DR were referred for in-person evaluation. Five of 11 patients with proliferative DR visited a University of Utah (U of U) ophthalmologist compared to 9 of 29 patients with moderate-severe DR. Four of 14 patients with advanced retinopathy seen by U of U ophthalmology began retinopathy-specific treatment (anti-VEGF injection/ photocoagulation). We conclude that initiation of point-of-care retinal screening to patients not routinely followed by ophthalmologists increased detection of mild, moderate and proliferative DR. These findings also identify follow-up with retinal specialists after referral as a potential treatment barrier. Disclosure M. Wahl: None. J. Neuberger: None. M. L. Litchman: Research Support; Self; Abbott Diabetes. G. S. Adjei-poku: None. A. Fagerlin: None. M. Hartnett: None. S. Fisher: None.

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 Disclosure R. Agrawal: None. S. Sharma: None. S. Fisher: None. Funding National Institutes of Health (R01NS070235-01A1, R01DK118082)

The brain has traditionally thought to be insensitive to insulin, primarily because insulin does not stimulate glucose uptake/metabolism in the brain (as it does in classic insulin sensitive tissues such as muscle, liver and fat). However, over the past 20 years, research in this field has identified unique actions of insulin in the brain. There is accumulating evidence that insulin crosses into the brain and regulates central nervous system functions such as feeding, depression and cognitive behavior. Additionally, insulin acts in the brain to regulate systemic functions such as hepatic glucose production, lipolysis, lipogenesis, reproductive competence and the sympathoadrenal response to hypoglycemia. Decrements in brain insulin action (or brain insulin resistance) can be observed in obesity, type 2 diabetes (T2DM), aging and Alzheimer's disease (AD), indicating a possible link between metabolic and cognitive health. Here, we describe recent findings on the pleiotropic actions of insulin in the brain and highlight the precise sites, specific neuronal population and roles for supportive astrocytic cells through which insulin acts in the brain. In addition, we also discuss how boosting brain insulin action could be a therapeutic option for people at an increased risk of developing metabolic and cognitive diseases such as AD and T2DM. Overall, this perspective article serves to highlight some of these key scientific findings, identify unresolved issues, and indicate future directions of research in this field that would serve to improve the lives of people with metabolic and cognitive dysfunctions.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes, and biochemical properties. Here, we report a fully active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy and protein engineering to elucidate its interactions with the human insulin receptor ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of native insulin but also compromise therapeutic utility by delaying the onset action, suggesting approaches to develop improved therapeutic insulins. Curiously, a receptor conformation present in low abundance adopts a highly asymmetric structure that displays novel coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions. [Figure not available: see fulltext.]

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 < 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 < 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)

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 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)

A cross-sectional observational cohort analysis was conducted to assess whether higher hypoglycemia alarm thresholds on CGMs are associated with less hypoglycemia for both patients with normal (NAH) and impaired (IAH) awareness of hypoglycemia. Two-week CGM alarm/glucose data from 39 and 42 type 1 diabetes (T1D) patients with NAH and IAH (determined by the Clarke questionnaire) who have used CGMs for >3 months and had an active CGM usage time of ≥86% were included in the analysis. IAH patients with higher hypoglycemia alarm thresholds (i.e., alarm thresholds set >73 mg/dL vs. Disclosure Y. Lin: None. D. Groat: None. O. Chan: None. R. Pop-Busui: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Consultant; Self; Bayer Healthcare Pharmaceuticals Inc., Novo Nordisk Inc. Research Support; Self; AstraZeneca. Other Relationship; Self; American Diabetes Association. M. Varner: None. S. Fisher: None. Funding National Institute for Diabetes and Digestive and Kidney Diseases/Washington University School of Medicine (2P30DK020579); University of Utah Diabetes and Metabolism Research Center; National Institute for Diabetes and Digestive and Kidney Diseases (5T32DK091317); National Center for Advancing Translational Sciences (1ULTR002538)

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone snail venom with moderate human-insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins—a human des-octapeptide insulin analog—as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a novel direction for therapeutic insulin development.

It is proposed that the impaired counterregulatory response (CRR) to hypoglycemia in insulin-deficient diabetes may be due to chronic brain insulin deficiency. To test this hypothesis, streptozotocin-induced diabetic Sprague-Dawley rats were infused with insulin (3 mU/day) or artificial cerebrospinal fluid (aCSF) bilaterally into the ventromedial hypothalamus (VMH) for 2 wk and compared with nondiabetic rats. Rats underwent hyperinsulinemic (50 mU·kg−1·min−1)-hypoglycemic (~45 mg/dl) clamps. Diabetic rats demonstrated an impaired CRR to hypoglycemia, noted by a high glucose infusion rate and blunted epinephrine and glucagon responses. The defective sympathoadrenal response was restored by chronic infusion of insulin into the VMH. Diabetic rats had decreased VMH Akt phosphorylation and decreased VMH glucose transporter 4 (GLUT4) content, which was also restored by chronic infusion of insulin into the VMH. Separate experiments in nondiabetic rats in which GLUT4 translocation into the VMH was inhibited with an infusion of indinavir were notable for an impaired CRR to hypoglycemia, indicated by increased glucose infusion rate and diminished epinephrine and glucagon responses. Results suggest that, in this model of diabetes, VMH insulin deficiency impairs the sympathoadrenal response to hypoglycemia and that chronic infusion of insulin into the VMH is sufficient to normalize the sympathoadrenal response to hypoglycemia via restoration of GLUT4 expression in the VMH.

Sulfonylureas increase the incidence of severe hypoglycemia in people with type 2 diabetes and might increase the risk of sudden cardiac death. Sulfonylureas stimulate insulin secretion by closing pancreatic ATP-sensitive potassium ion (KATP) channels. To investigate the role of KATP channel modulators on cardiac arrhythmias and mortality in the setting of severe hypoglycemia, adult Sprague-Dawley rats underwent hyperinsulinemic (0.2 U/kg/min) severe hypoglycemic (10 to 15 mg/dL) clamps with continuous electrocardiography. The rats were randomized for treatment with intravenous vehicle (VEH), the sulfonylurea glibenclamide (GLIB; KATP channel blocker; 5 mg/kg/h), or diazoxide (DIAZ; KATP channel opener; 5 mg/kg/h). The results demonstrated that GLIB completely prevented first-degree heart block compared with VEH (0.18 ± 0.09/min) and DIAZ (0.2 ± 0.05/min). Second-degree heart block was significantly reduced with GLIB (0.12 ± 0.1/min) compared with VEH (0.6 ± 0.2/min) and DIAZ (6.9 ± 3/min). The incidence of third-degree heart block was completely prevented by GLIB compared with VEH (67%) and DIAZ (87.5%). Hypoglycemia-induced mortality was completely prevented by GLIB compared with VEH (60%) and DIAZ (82%). In conclusion, although GLIB increases the risk of hypoglycemia by increasing insulin secretion, these results have demonstrated a paradoxical protective role of GLIB against severe hypoglycemia-induced fatal cardiac arrhythmias.

Supplemental Material

ContextLittle evidence exists regarding the positive and negative impacts of continuous glucose monitor system (CGM) alarm settings for diabetes control in patients with type 1 diabetes (T1D).ObjectiveEvaluate the associations between CGM alarm settings and glucose outcomes.Design and SettingA cross-sectional observational study in a single academic institution.Patients and Main Outcome MeasuresCGM alarm settings and 2-week CGM glucose information were collected from 95 T1D patients with > 3 months of CGM use and ≥ 86% active usage time. The associations between CGM alarm settings and glucose outcomes were analyzed.ResultsHigher glucose thresholds for hypoglycemia alarms (ie, ≥ 73 mg/dL vs < 73 mg/dL) were related to 51% and 65% less time with glucose < 70 and < 54 mg/dL, respectively (P = 0.005; P = 0.016), higher average glucose levels (P = 0.002) and less time-in-range (P = 0.005), but not more hypoglycemia alarms. The optimal alarm threshold for < 1% of time in hypoglycemia was 75 mg/dL.Lower glucose thresholds for hyperglycemia alarms (ie, ≤ 205 mg/dL vs > 205 mg/dL) were related to lower average glucose levels and 42% and 61% less time with glucose > 250 and > 320 mg/dL (P = 0.020, P = 0.016, P = 0.007, respectively), without more hypoglycemia. Lower alarm thresholds were also associated with more alarms (P < 0.0001). The optimal alarm threshold for < 5% of time in hyperglycemia and hemoglobin A1c ≤ 7% was 170 mg/dL.ConclusionsDifferent CGM glucose thresholds for hypo/hyperglycemia alarms are associated with various hypo/hyperglycemic outcomes. Configurations to the hypo/hyperglycemia alarm thresholds could be considered as an intervention to achieve therapeutic goals.

It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABA(A) receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3′-UTR of GABA(A) receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.

Hypoglycemia-associated autonomic failure (HAAF) is comprised of both a blunted counterregulatory response to hypoglycemia and hypoglycemia unawareness. We previously demonstrated that both these aspects of HAAF can be reversed by treatment with the dopamine receptor antagonist, metoclopramide. These findings suggest that repetitive activation of the dopaminergic system may contribute to HAAF. We now evaluate whether both the blunted counterregulatory response to hypoglycemia and hypoglycemia unawareness are mediated by repeated activation of brain dopaminergic receptors. To test this hypothesis, male Sprague-Dawley rats were randomized to receive either the dopamine receptor agonist, bromocriptine, (Bromo; 20 µg/d) intracerebroventricularly (ICV) for 3 consecutive days or recurrent ICV saline (controls). On the fourth day, all rats underwent either, 1) a hyperinsulinemic (50 mU/kg/min) hypoglycemic (∼50 mg/dl) clamp to assess counterregulation, or 2) had food consumption measured in response to insulin-induced (15 U/kg; SQ) hypoglycemia (∼40 mg/dl) to assess hypoglycemia awareness. In response to hypoglycemia, antecedent Bromo blunted, 1) the epinephrine response by 86%* (see Fig), 2) the glucagon response by 56%§, and 3) hypoglycemia awareness by 37%§, as compared to controls (*p Disclosure A. Vieira de Abreu: None. R. Agrawal: None. E.R. Meier: None. O. Chan: Research Support; Self; Zucara Therapeutics Inc. S. Fisher: None.

Based on the set glucose thresholds for hypoglycemia alarms, continuous glucose monitoring (CGM) systems can provide audible alerts that notify individuals for actual/impending hypoglycemic events. While CGMs have been shown to reduce hypoglycemic events in patients with type 1 diabetes (T1D), there are little data demonstrating the association between glucose thresholds for hypoglycemia alarms and clinical hypoglycemia outcomes. This cross-sectional observational study enrolled 83 T1D patients with CGM use >3 months, active CGM usage time ≥70% and activated (i.e., turned “on”) hypoglycemia alarms. CGM settings and two-week CGM glucose data (total 25,683 hours) were analyzed. Glucose thresholds for hypoglycemia alarms (mean: 74 ± 8 mg/dL; range: 60-100 mg/dL) correlated inversely with time in hypoglycemia (both glucose Disclosure Y. Lin: None. M. Hung: None. A. Sharma: None. O. Chan: Research Support; Self; Zucara Therapeutics Inc. M. Varner: None. D. Groat: None. D. Dopita: None. S. Fisher: None.

We tested the hypothesis that the impaired sympathoadrenal response induced by recurrent hypoglycemia (RH) is mediated by changes in key microRNA expression in the ventromedial hypothalamus (VMH). MicroRNA-sequencing data from the VMH of RH rats indicated a 67% downregulation of microRNA-7a-5p and a 2.3-fold upregulation of microRNA-665. Rescue experiments were conducted consisting of lentiviral-mediated overexpression of microRNA-7a-5p or downregulation of microRNA-665 targeted to the VMH. Rats were randomized to, 1) recurrent saline (RS), 2) RH (25-50 mg/dl x 3 days), 3) RH + Lenti-scrambled microRNA, or 4) RH + Lenti-targeted microRNA. All rats were subsequently subjected to hyperinsulinemic-hypoglycemic (40-50 mg/dl) clamps. As expected, RH induced a blunted sympathoadrenal hormone response to hypoglycemia. Targeted VMH overexpression (∼3.5 fold) of microRNA-7a-5p in RH rats normalized the epinephrine response to hypoglycemia coinciding with a 30% decreased glucose infusion rate. Targeted VMH downregulation of microRNA-665, however, did not improve counterreguatory responses. These findings indicate that microRNA-7a-5p is a potential mediator of impaired VMH glucose sensing/counterregulation and may be an effective therapeutic target to restore impaired sympathoadrenal responses to hypoglycemia. Disclosure R. Agrawal: None. M.G. Montgomery: None. D. Verma: None. G.T. Durupt: None. A. Vieira de Abreu: None. S. Swaminathan: None. S. Fisher: None.

Early detection and treatment of diabetic retinopathy (DR) can reduce blindness risk by >90%. Due to limitations in access to specialized eye care, the majority of people with diabetes (PWD) are not screened routinely. This project aims to determine if implementation of a point-of-care retinal screening program for PWD not routinely followed by ophthalmology would be effective in detecting DR in the Intermountain West region of the U.S. PWD who had not had a dilated eye exam within the last year underwent fundus photography during a regularly scheduled diabetes visit at the University of Utah Diabetes and Endocrinology Center. Non-mydriatic retinal images were classified by a vitreoretinal specialist as to DR severity based on the International Clinical Diabetic Retinopathy and Macular Edema Severity Scale. Abnormal findings were present in 49% of 116 patients imaged: Mild-moderate non-proliferative diabetic retinopathy (NPDR) in 16%, moderate-severe NPDR in 13%, proliferative retinopathy in 4% and hypertensive retinopathy in 13%. Point-of-care retinal imaging effectively identified DR and other retinal conditions in the Intermountain West. The finding of serious eye conditions in PWD not routinely followed by ophthalmologists supports the importance of improving ease of access for eye screening in diabetes care. Disclosure M. Wahl: None. A. Haynes: None. M.L. Litchman: None. A. Fagerlin: None. R.K. Delaney: None. J. Neuberger: None. G.S. Adjei-Poku: None. M. Serrato: None. M. Hartnett: None. S. Fisher: None.

In response to hypoglycemia, excessive stimulation of the parasympathetic nervous system (PNS) may induce bradycardia and fatal heart block. Since signaling via nicotinic receptors mediates the PNS response, it was hypothesized that these receptors may mediate hypoglycemia-induced cardiac arrhythmias. To test this hypothesis, mecamylamine (a nicotinic receptor antagonist; 7.5 mg/kg, n = 17) or saline (control; n = 20) was infused intravenously in Sprague Dawley rats during insulin-induced (0.2mU/kg/min) severe hypoglycemic (10-15 mg/dl) clamps for 3 hours with electrocardiogram recordings. Compared to controls, mecamylamine-treated rats required a 3-fold higher glucose infusion rate during severe hypoglycemia, consistent with lower peak epinephrine levels (5698±557 vs. 2418±396 pg/ml; p < 0.001). In control rats, hypoglycemia led to 2nd degree heart block (1.8±1.7/min), 3rd degree heart block (32%), and mortality (25%). However, mecamylamine treatment completely prevented 2nd and 3rd degree heart block resulting in 100% survival (*p < 0.05). In summary, blocking nicotinic receptors prevents cardiac arrhythmias and mortality during severe hypoglycemia. Clinically, targeting the parasympathetic nervous system could be a logical approach to prevent sudden death in people with insulin-treated diabetes at risk for hypoglycemia. Disclosure C.M. Reno: None. J. Bayles: None. Y. Huang: None. M.B. Oxspring: None. S. Fisher: None. Funding National Institutes of Health; National Institute of Diabetes and Digestive and Kidney Diseases; JDRF

Average glucose levels (AG) and glucose time-in-range (TIR; 70-180 mg/dL) are important glucose control measurements for patients with type 1 diabetes (T1D). Continuous glucose monitoring (CGM) systems provide audible alarms for actual/impending hyper/hypoglycemic events; however, there are little data regarding optimal CGM alarm settings to improve these measures. A cross-sectional observational study was conducted enrolling 91 T1D active CGM users (real-time CGM use >3 months and CGM usage time ≥70%). CGM alarm settings and two-week glucose data (total 28,133 hours) were analyzed. Glucose thresholds for hyperglycemia alarm and intra-threshold range (hyperglycemia minus hypoglycemia alarm thresholds) correlated positively with AG (P Disclosure Y. Lin: None. M. Hung: None. A. Sharma: None. O. Chan: Research Support; Self; Zucara Therapeutics Inc. M. Varner: None. D. Groat: None. D. Dopita: None. S. Fisher: None.

The contribution of the sympathetic nervous system (SNS) versus the parasympathetic nervous system (PSNS) in mediating fatal cardiac arrhythmias during insulin-induced severe hypoglycemia is not well understood. Therefore, experimental protocols were performed in nondiabetic Sprague-Dawley rats to test the SNS with 1) adrenal demedullation and 2) chemical sympathectomy, and to test the PSNS with 3) surgical vagotomy, 4) nicotinic receptor (mecamylamine) and muscarinic receptor (AQ-RA 741) blockade, and 5) ex vivo heart perfusions with normal or low glucose, acetylcholine (ACh), and/or mecamylamine. In protocols 1–4, 3-h hyperinsulinemic (0.2 units/kg/min) and hypoglycemic (10–15 mg/dL) clamps were performed. Adrenal demedullation and chemical sympathectomy had no effect on mortality or arrhythmias during severe hypoglycemia compared with controls. Vagotomy led to a 6.9-fold decrease in mortality; reduced first- and second-degree heart block 4.6- and 4-fold, respectively; and prevented third-degree heart block compared with controls. Pharmacological blockade of nicotinic receptors, but not muscarinic receptors, prevented heart block and mortality versus controls. Ex vivo heart perfusions demonstrated that neither low glucose nor ACh alone caused arrhythmias, but their combination induced heart block that could be abrogated by nicotinic receptor blockade. Taken together, ACh activation of nicotinic receptors via the vagus nerve is the primary mediator of severe hypoglycemia–induced fatal cardiac arrhythmias.

Objective: Impaired awareness of hypoglycemia (IAH) is a risk factor for severe hypoglycemia in patients with type 1 diabetes (T1D) not using a continuous glucose monitoring (CGM) system. The current study investigated the prevalence of IAH and its relationship with severe hypoglycemia in T1D patients using CGM systems. Methods: This cross-sectional observational study enrolled 135 patients with T1D and ongoing real-time CGM use. A survey was conducted to assess hypoglycemia awareness with the Gold, Clarke, and Pedersen-Bjergaard questionnaires and the 6-month history of severe hypoglycemia. Other diabetes histories and the CGM glucose data were collected. Results: The Gold, Clarke, and Pedersen-Bjergaard questionnaires demonstrated the overall prevalence of IAH/abnormal awareness to be 33.3%, 43.7%, and 77.0%, respectively. Participant age and duration of T1D were consistently related to IAH or hypoglycemia unawareness with all three questionnaires (P 6 months, 24.5% were found to have at least one episode of severe hypoglycemia in the preceding 6 months. IAH identified by the Gold and Clarke questionnaires and hypoglycemia unawareness identified by the Pedersen-Bjergaard questionnaire were related to 6-, 4.63-, and 5.83-fold increased risk of severe hypoglycemia (P = .001, .004, and .013), respectively. IAH identified by the Gold/Clarke questionnaires was associated with a longer duration of CGM glucose Conclusion: IAH is highly prevalent and related to a higher risk for severe hypoglycemia in T1D patients using CGM. Abbreviations: CGM = continuous glucose monitoring; CI = confidence interval; HAAF = hypoglycemia-associated autonomic failure; HbA1c = hemoglobin A1C; IAH = impaired awareness of hypoglycemia; T1D = type 1 diabetes

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.

AIMS/HYPOTHESIS: This study evaluates whether the non-selective β-blocker, carvedilol, can be used to prevent counterregulatory failure and the development of impaired awareness of hypoglycaemia (IAH) in recurrently hypoglycaemic rats. METHODS: Sprague Dawley rats were implanted with vascular catheters and intracranial guide cannulas targeting the ventromedial hypothalamus (VMH). These animals underwent either three bouts of insulin-induced hypoglycaemia or received three saline injections (control group) over 3 days. A subgroup of recurrently hypoglycaemic animals was treated with carvedilol. The next day, the animals underwent a hypoglycaemic clamp with microdialysis without carvedilol treatment to evaluate changes in central lactate and hormone levels. To assess whether carvedilol prevented IAH, we treated rats that had received repeated 2-deoxyglucose (2DG) injections to impair their awareness of hypoglycaemia with carvedilol and measured food intake in response to insulin-induced hypoglycaemia as a surrogate marker for hypoglycaemia awareness. RESULTS: Compared with the control group, recurrently hypoglycaemic rats had a ~1.7-fold increase in VMH lactate and this was associated with a 75% reduction in the sympathoadrenal response to hypoglycaemia. Treatment with carvedilol restored VMH lactate levels and improved the adrenaline (epinephrine) responses. In 2DG-treated rats compared with control animals receiving saline, food intake was reduced in response to hypoglycaemia and increased with carvedilol treatment. CONCLUSION/INTERPRETATION: We conclude that carvedilol may be a useful therapy to prevent counterregulatory failure and improve IAH.

Severe hypoglycemia causes fatal cardiac arrhythmias. The extent to which the parasympathetic nervous system regulates hypoglycemia-induced fatal arrhythmias remains unknown. To investigate the role of vagal innervation in regulating arrhythmias during severe hypoglycemia, Sprague Dawley rats underwent vagotomy (left vagus nerve transection adjacent to the carotid artery; n = 15) or sham surgery (control; n = 13). One week after surgery, all rats underwent hyperinsulinemic (0.2 U/kg/min) severe hypoglycemic (10-15 mg/dl) clamps with electrocardiogram recordings. Rats with vagotomy had increased heart rate at baseline and throughout the clamp, indicating successful vagotomy. Glucose infusion rates during hypoglycemia were increased 1.7-fold in rats with vagotomy, but epinephrine values were similar between the groups. Vagotomy resulted in fewer cardiac arrhythmias including a 12-fold decrease in 2nd degree heart block. Reduction in arrhythmias was associated with a 7-fold decrease in severe hypoglycemia-induced mortality in rats with vagotomy (7%) compared to controls (46%). In summary, vagal transection reduces mortality and cardiac arrhythmias during severe hypoglycemia. Since vagal innervation mediates severe hypoglycemia-induced sudden death, targeting the parasympathetic nervous system may be a logical approach to prevent sudden death for at-risk people with insulin treated diabetes. Disclosure C.M. Reno: None. A.J. Skinner: None. J. Bayles: None. S.J. Fisher: None.

We sought to develop a novel animal model of hypoglycemia-associated autonomic failure (HAAF) and investigate the pathological process involved. We previously noted that recurrent 2-deoxyglucose (R2DG) administration induced hypoglycemia unawareness (indicated by a blunted food intake response to hypoglycemia). We now evaluate whether R2DG also induces an impaired counterregulatory response and whether this pathological process is mediated by dopaminergic receptor activation. Sprague-Dawley rats were randomized to one of three, 3-day treatments: 1) recurrent saline (Control), 2) R2DG (200 mg/kg/d, SQ), or 3) R2DG and antecedent pretreatment with the dopaminergic antagonist metoclopramide (R2DG+Meto, 3 mg/kg/d, IP). On the fourth day, all rats underwent hyperinsulinemic (50 mU/kg/min) hypoglycemic (∼50 mg/dl) clamps. Notably, recurrent 2DG abrogated the epinephrine response to hypoglycemia as compared to controls. In addition to restoring hypoglycemia awareness (i.e., the food intake response to hypoglycemia), metoclopramide normalized the epinephrine response to hypoglycemia in 2DG treated rats. Glucagon showed a similar pattern of suppression with R2DG and restoration with R2DG+Meto. In this model of HAAF, our data identifies a novel role of dopaminergic receptor activation in the development of HAAF and a potential role for metoclopramide as a potential therapeutic agent to prevent HAAF. Disclosure A. Vieira de Abreu: None. R. Agrawal: None. P. Howe: None. S.J. Fisher: None.

We tested the hypothesis that the correction of ventromedial hypothalamus (VMH) insulin deficiency was sufficient to restore the impaired sympathoadrenal response to hypoglycemia in diabetic rats. Sprague-Dawley rats were injected with either vehicle (nondiabetic controls; CON) or streptozotocin (STZ; 65 mg/kg IP). STZ diabetic rats received osmotic mini-pumps to infuse either artificial cerebrospinal fluid (DIAB) or insulin (3mU/day; DIAB+InsVMH) into the VMH bilaterally. Two weeks later, all three groups underwent hyperinsulinemic (50 mU.kg-1.min-1) hypoglycemic (∼50 mg/dl) clamps. As expected, STZ-diabetic rats showed blunted epinephrine response to hypoglycemia as compared to nondiabetic controls. Chronic infusion of insulin into VMH of diabetic rats normalized the epinephrine response to hypoglycemia and lowered the glucose infusion rate required to maintain hypoglycemia. Examining brain insulin action, it was noted that STZ diabetic rats had decreased Akt phosphorylation and decreased expression of insulin dependent glucose transporter 4 (GLUT4) in VMH by 44 and 40% respectively as compared to the nondiabetic controls. Notably, chronic infusion of insulin to the VMH normalized Akt phosphorylation and GLUT4 expression. In summary, insulin acts chronically in the VMH to preserve the sympathoadrenal response to hypoglycemia, possibly by regulating GLUT4 expression. Disclosure R. Agrawal: None. A. Vieira de Abreu: None. G.T. Durupt: None. S.J. Fisher: None.

We previously demonstrated that insulin-induced severe hypoglycemia-associated sudden death is largely mediated by fatal cardiac arrhythmias. In the current study, a pharmacological approach was taken to explore the potential contribution of hypoglycemic seizures and the sympathoadrenergic system in mediating severe hypoglycemia-associated sudden death. Adult Sprague-Dawley rats were randomized into one of four treatment groups: 1) saline (SAL), 2) anti-arrhythmic (β1 blocker atenolol), 3) antiseizure (levetiracetam), and 4) combination antiarrhythmic and antiseizure (β1 Blocker+Levetiracetam). All rats underwent hyperinsulinemic severe hypoglycemic clamps for 3.5 h. When administered individually during severe hypoglycemia, β1 blocker reduced 2nd and 3rd degree heart block by 7.7- and 1.6-fold, respectively, and levetiracetam reduced seizures 2.7-fold, but mortality in these groups did not decrease. However, it was combined treatment with both β1 blocker and levetiracetam that remarkably reduced seizures and completely prevented respiratory arrest, while also eliminating 2nd and 3rd degree heart block, leading to 100% survival. These novel findings demonstrate that, in mediating sudden death, hypoglycemia elicits two distinct pathways (seizure-associated respiratory arrest and arrhythmia-associated cardiac arrest), and therefore, prevention of both seizures and cardiac arrhythmias is necessary to prevent severe hypoglycemia-induced mortality.

People with insulin-treated diabetes are uniquely at risk for severe hypoglycemia-induced brain damage. Because calcium influx may mediate brain damage, we tested the hypothesis that the calcium-channel blocker, verapamil, would significantly reduce brain damage and cognitive impairment caused by severe hypoglycemia. Sprague-Dawley rats (10 weeks old) were randomly assigned to one of three treatments: 1) control hyperinsulinemic (200 mU ⋅ kg−1 ⋅ min−1)-euglycemic (80–100 mg/dL) clamps (n = 14), 2) hyperinsulinemic-hypoglycemic (10–15 mg/dL) clamps (n = 16), or 3) hyperinsulinemic-hypoglycemic clamps, followed by a single treatment with verapamil (20 mg/kg) (n = 11). Compared with euglycemic controls, hypoglycemia markedly increased dead/dying neurons in the hippocampus by 16-fold and cortex by 14-fold. Verapamil treatment strikingly decreased hypoglycemia-induced hippocampal and cortical damage, by 87% and 94%, respectively. Morris Water Maze probe trial results demonstrated that hypoglycemia induced a retention, but not encoding, memory deficit (noted by both abolished target quadrant preference and reduced target quadrant time). Verapamil treatment significantly rescued spatial memory as noted by restoration of target quadrant preference and target quadrant time. In summary, a one-time treatment with verapamil after severe hypoglycemia prevented neural damage and memory impairment caused by severe hypoglycemia. For people with insulin-treated diabetes, verapamil may be a useful drug to prevent hypoglycemia-induced brain damage.

We previously demonstrated that insulin-mediated severe hypoglycemia induces lethal cardiac arrhythmias. However, whether chronic diabetes and insulin deficiency exacerbates, and whether recurrent antecedent hypoglycemia ameliorates, susceptibility to arrhythmias remains unknown. Thus, adult Sprague-Dawley rats were randomized into four groups: 1) nondiabetic (NONDIAB), 2) streptozotocin-induced insulin deficiency (STZ), 3) STZ with antecedent recurrent (3 days) hypoglycemia (∼40–45 mg/dL, 90 min) (STZ+RH), and 4) insulin-treated STZ (STZ+Ins). Following treatment protocols, all rats underwent hyperinsulinemic (0.2 units ⋅ kg−1 ⋅ min−1), severe hypoglycemic (10–15 mg/dL) clamps for 3 h with continuous electrocardiographic recordings. During matched nadirs of severe hypoglycemia, rats in the STZ+RH group required a 1.7-fold higher glucose infusion rate than those in the STZ group, consistent with the blunted epinephrine response. Second-degree heart block was increased 12- and 6.8-fold in the STZ and STZ+Ins groups, respectively, compared with the NONDIAB group, yet this decreased 5.4-fold in the STZ+RH group compared with the STZ group. Incidence of third-degree heart block in the STZ+RH group was 5.6%, 7.8-fold less than the incidence in the STZ group (44%). Mortality due to severe hypoglycemia was 5% in the STZ+RH group, 6.2-fold less than that in the STZ group (31%). In summary, severe hypoglycemia–induced cardiac arrhythmias were increased by insulin deficiency and diabetes and reduced by antecedent recurrent hypoglycemia. In this model, recurrent moderate hypoglycemia reduced fatal severe hypoglycemia–induced cardiac arrhythmias.

For people with diabetes, hypoglycemia remains the limiting factor in achieving glycemic control. This article reviews recent advances in how the brain senses and responds to hypoglycemia. Novel mechanisms by which individuals with insulin-treated diabetes develop hypoglycemia unawareness and impaired counterregulatory responses are outlined. Prevention strategies for reducing the incidence of hypoglycemia are discussed.

Hypoglycemia is a common complication for insulin treated people with diabetes. Severe hypoglycemia, which occurs in the setting of excess or ill-timed insulin administration, has been shown to cause brain damage. Previous pre-clinical studies have shown that memantine (an N-methyl-d-aspartate receptor antagonist) and erythropoietin can be neuroprotective in other models of brain injury. We hypothesized that these agents might also be neuroprotective in response to severe hypoglycemia-induced brain damage. To test this hypothesis, 9-week old, awake, male Sprague-Dawley rats underwent hyperinsulinemic (0.2 U kg(-1)min(-1)) hypoglycemic clamps to induce severe hypoglycemia (blood glucose 10-15 mg/dl for 90 min). Animals were randomized into control (vehicle) or pharmacological treatments (memantine or erythropoietin). One week after severe hypoglycemia, neuronal damage was assessed by Fluoro-Jade B and hematoxylin and eosin staining of brain sections. Treatment with both memantine and erythropoietin significantly decreased severe hypoglycemia-induced neuronal damage in the cortex by 35% and 39%, respectively (both p0.05 vs. controls). These findings demonstrate that memantine and erythropoietin provide a protective effect against severe hypoglycemia-induced neuronal damage.

OBJECTIVE An impaired ability to sense and appropriately respond to insulin-induced hypoglycemia is a common and serious complication faced by insulin-treated diabetic patients. This study tests the hypothesis that insulin acts directly in the brain to regulate critical glucose-sensing neurons in the hypothalamus to mediate the counterregulatory response to hypoglycemia. RESEARCH DESIGN AND METHODS To delineate insulin actions in the brain, neuron-specific insulin receptor knockout (NIRKO) mice and littermate controls were subjected to graded hypoglycemic (100, 70, 50, and 30 mg/dl) hyperinsulinemic (20 mU/kg/min) clamps and nonhypoglycemic stressors (e.g., restraint, heat). Subsequently, counterregulatory responses, hypothalamic neuronal activation (with transcriptional marker c-fos), and regional brain glucose uptake (via 14C-2deoxyglucose autoradiography) were measured. Additionally, electrophysiological activity of individual glucose-inhibited neurons and hypothalamic glucose sensing protein expression (GLUTs, glucokinase) were measured. RESULTS NIRKO mice revealed a glycemia-dependent impairment in the sympathoadrenal response to hypoglycemia and demonstrated markedly reduced (3-fold) hypothalamic c-fos activation in response to hypoglycemia but not other stressors. Glucose-inhibited neurons in the ventromedial hypothalamus of NIRKO mice displayed significantly blunted glucose responsiveness (membrane potential and input resistance responses were blunted 66 and 80%, respectively). Further, hypothalamic expression of the insulin-responsive GLUT 4, but not glucokinase, was reduced by 30% in NIRKO mice while regional brain glucose uptake remained unaltered. CONCLUSIONS Chronically, insulin acts in the brain to regulate the counterregulatory response to hypoglycemia by directly altering glucose sensing in hypothalamic neurons and shifting the glycemic levels necessary to elicit a normal sympathoadrenal response.

OBJECTIVE Although intensive glycemic control achieved with insulin therapy increases the incidence of both moderate and severe hypoglycemia, clinical reports of cognitive impairment due to severe hypoglycemia have been highly variable. It was hypothesized that recurrent moderate hypoglycemia preconditions the brain and protects against damage caused by severe hypoglycemia. RESEARCH DESIGN AND METHODS Nine-week-old male Sprague-Dawley rats were subjected to either 3 consecutive days of recurrent moderate (25–40 mg/dl) hypoglycemia (RH) or saline injections. On the fourth day, rats were subjected to a hyperinsulinemic (0.2 units · kg−1 · min−1) severe hypoglycemic (∼11 mg/dl) clamp for 60 or 90 min. Neuronal damage was subsequently assessed by hematoxylin-eosin and Fluoro-Jade B staining. The functional significance of severe hypoglycemia–induced brain damage was evaluated by motor and cognitive testing. RESULTS Severe hypoglycemia induced brain damage and striking deficits in spatial learning and memory. Rats subjected to recurrent moderate hypoglycemia had 62–74% less brain cell death and were protected from most of these cognitive disturbances. CONCLUSIONS Antecedent recurrent moderate hypoglycemia preconditioned the brain and markedly limited both the extent of severe hypoglycemia–induced neuronal damage and associated cognitive impairment. In conclusion, changes brought about by recurrent moderate hypoglycemia can be viewed, paradoxically, as providing a beneficial adaptive response in that there is mitigation against severe hypoglycemia–induced brain damage and cognitive dysfunction.

Insulin-induced severe hypoglycemia causes brain damage. The hypothesis to be tested was that diabetes portends to more extensive brain tissue damage following an episode of severe hypoglycemia. Nine-week-old male streptozotocin-diabetic (DIAB; n = 10) or vehicle-injected control (CONT; n = 7) Sprague-Dawley rats were subjected to hyperinsulinemic (0.2 U·kg−1·min−1) severe hypoglycemic (10–15 mg/dl) clamps while awake and unrestrained. Groups were precisely matched for depth and duration (1 h) of severe hypoglycemia (CONT 11 ± 0.5 and DIAB 12 ± 0.2 mg/dl, P = not significant). During severe hypoglycemia, an equal number of episodes of seizure-like activity were noted in both groups. One week later, histological analysis demonstrated extensive neuronal damage in regions of the hippocampus, especially in the dentate gyrus and CA1 regions and less so in the CA3 region ( P < 0.05), although total hippocampal damage was not different between groups. However, in the cortex, DIAB rats had significantly (2.3-fold) more dead neurons than CONT rats ( P < 0.05). There was a strong correlation between neuronal damage and the occurrence of seizure-like activity ( r2 > 0.9). Separate studies conducted in groups of diabetic ( n = 5) and nondiabetic ( n = 5) rats not exposed to severe hypoglycemia showed no brain damage. In summary, under the conditions studied, severe hypoglycemia causes brain damage in the cortex and regions within the hippocampus, and the extent of damage is closely correlated to the presence of seizure-like activity in nonanesthetized rats. It is concluded that, in response to insulin-induced severe hypoglycemia, diabetes uniquely increases the vulnerability of specific brain areas to neuronal damage.

This study tests the hypothesis that lipids could act as an alternative fuel source in the brain during insulin-induced hypoglycemia. Male Sprague-Dawley rats were subjected to hyperinsulinemic (5 mU·kg−1·min−1) hypoglycemic (∼50 mg/dl) clamps. In protocol 1, intralipid (IL), a fat emulsion, was infused intravenously to prevent the fall in free fatty acid levels that occurs in response to hyperinsulinemic hypoglycemia. Intravenous lipid infusion did not alter the counterregulatory responses to hypoglycemia. To test whether IL could have central effects in mediating the counterregulatory response to hypoglycemia, in protocol 2 the brains of precannulated rats were intracerebroventricularly (icv) infused with IL or artificial cerebrospinal fluid (aCSF) as control. Unexpectedly, the epinephrine and glucagon response to hypoglycemia was significantly augmented with icv IL infusion. To determine whether central IL infusion could restore defective counterregulation, in protocol 3 rats were made recurrently hypoglycemic (RH) for 3 days and on the 4th day underwent hyperinsulinemic hypoglycemic clamps with icv IL or aCSF infusion. RH rats had the expected impaired epinephrine response to hypoglycemia, and icv IL infusion again significantly augmented the epinephrine response in RH rats to normal. With regard to our experimental model of hypoglycemic counterregulation, we conclude that 1) systemic lipid infusion did not alter the counterregulatory response to hypoglycemia, 2) the icv infusion of lipids markedly increased CSF FFA levels and paradoxically augmented the epinephrine and glucagon responses, and 3) the blunted sympathoadrenal response in recurrently hypoglycemic rats was completely normalized with the icv lipid infusion. It is concluded that, in the setting of insulin-induced hypoglycemia, increased brain lipids can enhance the sympathoadrenal response.

Hypoglycemia, hypoglycemia unawareness, and impaired counterregulation are major challenges to the intensive management of type 1 diabetes. While the counterregulatory response to hypoglycemia is predominantly determined by the degree and duration of hypoglycemia, there is now evidence that insulin per se may influence the counterregulatory response to hypoglycemia. To define the role of insulin action in the central nervous system in regulating the counterregulatory response to hypoglycemia, mice with a brain/neuron-specific insulin receptor knockout (NIRKO) and littermate controls were subjected to 90-min hyperinsulinemic (20 mU x kg(-1) x min(-1)) -hypoglycemic (approximately 1.5 mmol/l) clamps. In response to hypoglycemia, epinephrine levels rose 5.7-fold in controls but only 3.5-fold in NIRKO mice. Similarly, in response to hypoglycemia, norepinephrine levels rose threefold in controls, but this response was almost completely absent in NIRKO mice. In contrast, glucagon and corticosterone responses to hypoglycemia were similar in both groups. Thus, insulin action in the brain is critical for full activation of the sympathoadrenal response to hypoglycemia, and altered neural insulin signaling could contribute to defective glucose counterregulation in diabetes.

Low birth weight (LBW) is an important risk factor for type 2 diabetes. We have developed a mouse model of LBW resulting from undernutrition during pregnancy. Restriction of maternal food intake from day 12.5 to 18.5 of pregnancy results in a 23% decrease in birth weight (P < 0.001), with normalization after birth. However, offspring of undernutrition pregnancies develop progressive, severe glucose intolerance by 6 months. To identify early defects that are responsible for this phenotype, we analyzed mice of undernutrition pregnancies at age 2 months, before the onset of glucose intolerance. Fed insulin levels were 1.7-fold higher in mice of undernutrition pregnancies (P = 0.01 vs. controls). However, insulin sensitivity was normal in mice of undernutrition pregnancies, with normal insulin tolerance, insulin-stimulated glucose disposal, and isolated muscle and adipose glucose uptake. Although insulin clearance was mildly impaired in mice of undernutrition pregnancies, the major metabolic phenotype in young mice of undernutrition pregnancies was dysregulation of insulin secretion. Despite normal β-cell mass, islets from normoglycemic mice of undernutrition pregnancies showed basal hypersecretion of insulin, complete lack of responsiveness to glucose, and a 2.5-fold increase in hexokinase activity. Taken together, these data suggest that, at least in mice, primary β-cell dysfunction may play a significant role in the pathogenesis of LBW-associated type 2 diabetes.