Hypoglycemic Counterregulation Research Articles

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.

An evolving spectrum of diabetes in a woman with GCK-MODY.

Coexistence of autoimmune diabetes and maturity-onset diabetes of the young (MODY) is rare. We report the first case of coexisting latent autoimmune diabetes of adulthood (LADA) and glucokinase (GCK) MODY. A 32-year-old woman was treated with insulin for gestational diabetes at age 32 years; post-partum, her fasting blood glucose was 6.0 mmol/L and 2-h glucose was 11.8 mmol/L following an oral glucose tolerance test, and she was maintained on diet alone. Five years later, a diagnosis of LADA was made when she presented with fasting blood glucose of 20.3 mmol/L and HbA1C 125 mmol/mol (13.6%). GCK-MODY was identified 14 years later when genetic testing was prompted by identification of a mutation in her cousin. Despite multiple daily insulin injections her glycaemic control remained above target and her clinical course has been complicated by multiple episodes of hypoglycaemia with unawareness. Although rare, coexistence of latent autoimmune diabetes of adulthood and monogenic diabetes should be considered if there is a strong clinical suspicion, for example, family history. Hypoglycaemic unawareness developed secondary to frequent episodes of hypoglycaemia using standard glycaemic targets for LADA. This case highlights the importance of setting fasting glucose targets within the expected range for GCK-MODY in subjects with coexisting LADA. Learning points: We report the first case of coexisting latent autoimmune diabetes of adulthood (LADA) and GCK-MODY. It has been suggested that mutations in GCK may lead to altered counter-regulation and recognition of hypoglycaemia at higher blood glucose levels than patients without such mutation. However, in our case, hypoglycaemic unawareness developed secondary to frequent episodes of hypoglycaemia using standard glycaemic targets for LADA. This case highlights the importance of setting fasting glucose targets within the expected range for GCK-MODY in subjects with coexisting LADA to avoid hypoglycaemia.

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

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

Hypoglycaemia and gastric emptying.

Hypoglycaemia is arguably the most important complication of insulin therapy in type 1 and type 2 diabetes. Counter-regulation of hypoglycaemia is dependent on autonomic function and frequent hypoglycaemia may lead to reductions in both autonomic warning signals and the catecholamine response, the so-called "impaired awareness of hypoglycaemia". It is now appreciated that gastric emptying is a major determinant of the glycaemic response to carbohydrate-containing meals in both health and diabetes, that disordered (especially delayed) gastric emptying occurs frequently in diabetes, and that acute hypoglycaemia accelerates gastric emptying substantially. However, the potential relevance of gastric emptying to the predisposition to, and counter-regulation of, hypoglycaemia has received little attention. In insulin-treated patients, the rate of gastric emptying influences the timing of the postprandial insulin requirement, and gastroparesis is likely to predispose to postprandial hypoglycaemia. Conversely, the marked acceleration of gastric emptying induced by hypoglycaemia probably represents an important counter-regulatory response to increase the rate of carbohydrate absorption. This review summarizes the current knowledge of the inter-relationships between hypoglycaemia and gastric emptying, with a focus on clinical implications.

Hypothalamic POMC or MC4R deficiency impairs counterregulatory responses to hypoglycemia in mice

ObjectiveLife-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation. MethodsTo test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes. ResultsStimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice. ConclusionIn conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes.

Acute Caloric Restriction Leads to Loss of Hypoglycemic Counter-Regulation in Mice following Short-Term Refeeding

Background: Hypoglycemia-associated autonomic failure (HAAF) is a maladaptive failure in glucose counter-regulation known to be caused by recurrent exposure to insulin-induced hypoglycemia. Fasting or caloric restriction is the only natural physiological process which induces prolonged moderate-to-severe hypoglycemia similar to the antecedent of HAAF. In this study we tested the hypothesis that exposure to fasting-induced hypoglycemia can cause HAAF-like symptoms in mice. Methods: Two groups of mice were used, an ad libitum (ad lib) fed group (n=6) and a caloric restriction (CR) group (n=6). 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 (1.4-1.75 U/kg, IP) which produced similar levels of hypoglycemia in each group (∼45 mg/dL). Results: As expected, mice exposed to 6 days of 60% CR lost ≈30% of their initial body weight, and experienced a gradual fall in blood glucose to an average minimum of 67.9 mg/dL. CR mice displayed a significantly reduced fasting glucose level relative to ad lib mice (102.7 and 171.0 mg/dL respectively, p = 0.0002) as well as reduced serum glucagon levels in response to the ITT (6.0 and 30.4 pg/mL respectively, p = 0.0182). Conclusions: Our results demonstrate that exposure to fasting-induced hypoglycemia produces HAAF-like symptoms in mice following refeeding. These results suggest that HAAF may be a result of an adaptive response to fasting which is inappropriately elicited during exposure to insulin-induced hypoglycemia. Further characterization of the effects of CR on the induction of deficits in glucose counterregulation warrants further investigation. Disclosure D. McDougal: None.

Hypoglycemia: Role of Hypothalamic Glucose-Inhibited (GI) Neurons in Detection and Correction.

Hypoglycemia is a profound threat to the brain since glucose is its primary fuel. As a result, glucose sensors are widely located in the central nervous system and periphery. In this perspective we will focus on the role of hypothalamic glucose-inhibited (GI) neurons in sensing and correcting hypoglycemia. In particular, we will discuss GI neurons in the ventromedial hypothalamus (VMH) which express neuronal nitric oxide synthase (nNOS) and in the perifornical hypothalamus (PFH) which express orexin. The ability of VMH nNOS-GI neurons to depolarize in low glucose closely parallels the hormonal response to hypoglycemia which stimulates gluconeogenesis. We have found that nitric oxide (NO) production in low glucose is dependent on oxidative status. In this perspective we will discuss the potential relevance of our work showing that enhancing the glutathione antioxidant system prevents hypoglycemia associated autonomic failure (HAAF) in non-diabetic rats whereas VMH overexpression of the thioredoxin antioxidant system restores hypoglycemia counterregulation in rats with type 1 diabetes.We will also address the potential role of the orexin-GI neurons in the arousal response needed for hypoglycemia awareness which leads to behavioral correction (e.g., food intake, glucose administration). The potential relationship between the hypothalamic sensors and the neurocircuitry in the hindbrain and portal mesenteric vein which is critical for hypoglycemia correction will then be discussed.

Acute Hypoglycemia in Healthy Humans Impairs Insulin-Stimulated Glucose Uptake and Glycogen Synthase in Skeletal Muscle: A Randomized Clinical Study.

Hypoglycemia is the leading limiting factor in glycemic management of insulin-treated diabetes. Skeletal muscle is the predominant site of insulin-mediated glucose disposal. Our study used a crossover design to test to what extent insulin-induced hypoglycemia affects glucose uptake in skeletal muscle and whether hypoglycemia counterregulation modulates insulin and catecholamine signaling and glycogen synthase activity in skeletal muscle. Nine healthy volunteers were examined on three randomized study days: 1) hyperinsulinemic hypoglycemia (bolus insulin), 2) hyperinsulinemic euglycemia (bolus insulin and glucose infusion), and 3) saline control with skeletal muscle biopsies taken just before, 30 min after, and 75 min after insulin/saline injection. During hypoglycemia, glucose levels reached a nadir of ∼2.0 mmol/L, and epinephrine rose to ∼900 pg/mL. Hypoglycemia impaired insulin-stimulated glucose disposal and glucose clearance in skeletal muscle, whereas insulin signaling in glucose transport was unaffected by hypoglycemia. Insulin-stimulated glycogen synthase activity was completely ablated during hyperinsulinemic hypoglycemia, and catecholamine signaling via cAMP-dependent protein kinase and phosphorylation of inhibiting sites on glycogen synthase all increased.

Ventromedial hypothalamic glucose sensing and glucose homeostasis vary throughout the estrous cycle

Objective17β-Estradiol (17βE) regulates glucose homeostasis in part by centrally mediated mechanisms. In female rodents, the influence of the ovarian cycle on hypoglycemia counterregulation and glucose tolerance is unclear. We found previously that in prepubertal females, 17βE modulates glucose sensing in nonadapting glucose-inhibited (GI) and adapting GI (AdGI) neurons within the ventrolateral portion of the ventromedial nucleus (VL-VMN). Nonadapting GI neurons persistently decrease their activity as glucose increases while AdGI neurons transiently respond to a glucose increase. To begin to understand if endogenous fluctuations in estrogen levels across the estrous cycle impact hypothalamic glucose sensing and glucose homeostasis, we assessed whether hypoglycemia counterregulation and glucose tolerance differed across the phases of the estrous cycle. We hypothesized that the response to insulin-induced hypoglycemia (IIH) and/or glucose tolerance would vary throughout the estrous cycle according to changes in 17βE availability. Moreover, that these changes would correlate with estrous-dependent changes in the glucose sensitivity of VL-VMN glucose-sensing neurons (GSNs). MethodsThese hypotheses were tested in female mice by measuring the response to IIH, glucose tolerance and the glucose sensitivity of VL-VMN GSNs during each phase of the estrous cycle. Furthermore, a physiological brain concentration of 17βE seen during proestrus was acutely applied to brain slices isolated on the day of diestrous and the response to low glucose in VL-VMN GSNs was assayed. ResultsThe response to IIH was strongest during diestrous. The response of nonadapting GI and AdGI neurons to a glucose decrease from 2.5 to 0.5mM also peaked during diestrous; an effect which was blunted by the addition of 17βE. In contrast, the glucose sensitivity of the subpopulation of GSNs which are excited by glucose (GE) was not affected by estrous phase or exogenous 17βE application. ConclusionThese data suggest that physiological fluctuations in circulating 17βE levels across the estrous cycle lead to changes in hypothalamic glucose sensing and the response to IIH.

Spontaneous Hypoglycemia After Islet Transplantation: The Case For Using Non-Hepatic Sites.

This Perspective provides a brief history of intrahepatic alloislet and autoislet transplantation in humans and an update of the recent success rates. It also examines the important role that hypoglycemia plays in clinical outcomes. On the one hand, recurrent serious hypoglycemic episodes related to insulin therapy are a major criterion for alloislet transplantation. On the other hand, spontaneous clinical hypoglycemia, perhaps related to the accompanying Roux-en-Y procedure for total pancreatectomy, is a complication of autoislet transplantation. Complex alterations in glucagon secretion compromise counter-regulation of hypoglycemia in both situations. The glucagon response to hypoglycemia is intrinsically defective in type 1 diabetes before transplant because of the absence of physiological regulation of α-cell secretion by neighboring β-cells. Glucagon secretion from intrahepatic islets during systemic hypoglycemia is also defective, although β-cells in the graft are normally regulated by glucose and arginine. My personal perspective is that the latter is caused by intrahepatic glycogenolysis stimulated by systemic hypoglycemia with consequent increases in intrahepatic glucose flux, which incorrectly signals intrahepatic α-cells to be quiescent. This defect is liver-specific, which strongly suggests modifying the current approach to islet transplantation by placing a portion of allo- and autoislets in nonhepatic sites in addition to hepatic sites to ensure physiological glucagon secretion as a strategy to ameliorate post-transplant hypoglycemia.

Hepatic glycogen can regulate hypoglycemic counterregulation via a liver-brain axis.

Liver glycogen is important for the counterregulation of hypoglycemia and is reduced in individuals with type 1 diabetes (T1D). Here, we examined the effect of varying hepatic glycogen content on the counterregulatory response to low blood sugar in dogs. During the first 4 hours of each study, hepatic glycogen was increased by augmenting hepatic glucose uptake using hyperglycemia and a low-dose intraportal fructose infusion. After hepatic glycogen levels were increased, animals underwent a 2-hour control period with no fructose infusion followed by a 2-hour hyperinsulinemic/hypoglycemic clamp. Compared with control treatment, fructose infusion caused a large increase in liver glycogen that markedly elevated the response of epinephrine and glucagon to a given hypoglycemia and increased net hepatic glucose output (NHGO). Moreover, prior denervation of the liver abolished the improved counterregulatory responses that resulted from increased liver glycogen content. When hepatic glycogen content was lowered, glucagon and NHGO responses to insulin-induced hypoglycemia were reduced. We conclude that there is a liver-brain counterregulatory axis that is responsive to liver glycogen content. It remains to be determined whether the risk of iatrogenic hypoglycemia in T1D humans could be lessened by targeting metabolic pathway(s) associated with hepatic glycogen repletion.

Leptin acts in the brain to influence hypoglycemic counterregulation: disparate effects of acute and recurrent hypoglycemia on glucagon release.

Leptin has been shown to diminish hyperglycemia via reduced glucagon secretion, although it can also enhance sympathoadrenal responses. However, whether leptin can also inhibit glucagon secretion during insulin-induced hypoglycemia or increase epinephrine during acute or recurrent hypoglycemia has not been examined. To test whether leptin acts in the brain to influence counterregulation, hyperinsulinemic hypoglycemic (∼45 mg/dl) clamps were performed on rats exposed to or not exposed to recurrent hypoglycemia (3 days, ∼40 mg/dl). Intracerebroventricular artificial cerebral spinal fluid or leptin was infused during the clamp. During acute hypoglycemia, leptin decreased glucagon responses by 51% but increased epinephrine and norepinephrine by 24 and 48%, respectively. After recurrent hypoglycemia, basal plasma leptin levels were undetectable. Subsequent brain leptin infusion during hypoglycemia paradoxically increased glucagon by 45% as well as epinephrine by 19%. In conclusion, leptin acts within the brain to diminish glucagon secretion during acute hypoglycemia but increases epinephrine, potentially limiting its detrimental effects during hypoglycemia. Exposure to recurrent hypoglycemia markedly suppresses plasma leptin, whereas exogenous brain leptin delivery enhances both glucagon and epinephrine release to subsequent hypoglycemia. These data suggest that recurrent hypoglycemia may diminish counterregulatory responses in part by reducing brain leptin action.

Free fatty acid availability is closely related to myocardial lipid storage and cardiac function in hypoglycemia counterregulation.

Hypoglycemia, a major side effect of intensive glucose-lowering therapy, was recently linked to increased cardiovascular risk in patients with diabetes. Whether increased circulating free fatty acids (FFA) owing to catecholamine-induced lipolysis affect myocardial energy metabolism and thus link hypoglycemia to cardiac vulnerability is unclear. Therefore, this study investigated the impact of hypoglycemia counterregulation (± inhibition of lipolysis) on myocardial lipid content (MYCL) and left ventricular function in healthy subjects. Nine healthy men were studied in randomized order: 1) insulin/hypoglycemia test (IHT; ins+/aci-), 2) IHT during inhibition of adipose tissue lipolysis by acipimox (ins+/aci+), 3) normoglycemia with acipimox (ins-/aci+), and 4) normoglycemia with placebo (ins-/aci-). MYCL and cardiac function were assessed by employing magnetic resonance spectroscopy/imaging at baseline and at 2 and 6 h. In response to acute hypoglycemia, plasma FFA (P<0.0001) and ejection fraction (EF; from 63.2±5.5 to 69.6±6.3%, P=0.0001) increased significantly and were tightly correlated with each other (r=0.68, P=0.0002); this response was completely blunted by inhibition of adipose tissue lipolysis. In the presence of normoglycemia, inhibition of lipolysis was associated with a drop in EF (from 59.2±5.5 to 53.9±6.9%,P=0.005) and a significant decrease in plasma FFA, triglycerides, and MYCL (by 48.5%, P=0.0001). The present data indicate that an intact interorgan cross-talk between adipose tissue and the heart is a prerequisite for catecholamine-mediated myocardial contractility and preservation of myocardial lipid stores in response to acute hypoglycemia.

Effect of bilateral carotid body resection on the counterregulatory response to hypoglycaemia in humans.

What is the central question of this study? Hyperoxia blunts hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in carotid body-resected patients; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. What is the main finding and its importance? Our data indicate that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, a relatively normal counterregulatory response to hypoglycaemia in carotid body-resected patients highlights: (i) the potential for long-term adaptations after carotid body resection; and (ii) the importance of redundant mechanisms in mediating hypoglycaemia counterregulation. Hyperoxia reduces hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in patients with bilateral carotid body resection; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. Five patients (three male and two female) with bilateral carotid body resection for glomus tumours underwent two 180 min hyperinsulinaemic, hypoglycaemic (∼ 3.3 mmol l(-1)) clamps separated by a minimum of 1 week and randomized to either normoxia (21% fractional inspired O2 ) or hyperoxia (100% fractional inspired O2). Ten healthy adults (seven male and three female) served as control subjects. Hypoglycaemia counterregulation in carotid body-resected patients was not significantly altered by hyperoxia (area under the curve expressed as a percentage of the normoxic response: glucose infusion rate, 111 ± 10%; cortisol, 94 ± 6%; glucagon, 107 ± 7%; growth hormone, 92 ± 10%; adrenaline, 89 ± 26%; noradrenaline, 79 ± 15%; main effect of condition, P > 0.05). This is in contrast to previously published results from healthy adults. However, the counterregulatory responses to hypoglycaemia during normoxia were not impaired in carotid body-resected patients when compared with control subjects (main effect of group, P > 0.05). Our data provide further corroborative evidence that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.

Peripheral and central glucose sensing in hypoglycemic detection.

Hypoglycemia poses a serious threat to the integrity of the brain, owing to its reliance on blood glucose as a fuel. Protecting against hypoglycemia is an extended network of glucose sensors located within the brain and in the periphery that serve to mediate responses restoring euglycemia, i.e., counterregulatory responses. This review examines the various glucose sensory loci involved in hypoglycemic detection, with a particular emphasis on peripheral glucose sensory loci and their contribution to hypoglycemic counterregulation.

Essentiality of Portal Vein Receptors in Hypoglycemic Counterregulation: Direct Proof Via Denervation in Male Canines

A major issue of in the treatment of diabetes is the risk of hypoglycemia. Hypoglycemia is detected both centrally and peripherally in the porto-hepatic area. The portal locus for hypoglycemic detection was originally described using the "local irrigation of the liver" approach in a canine model. Further work using portal vein denervation (DEN) in a rodent model characterized portal hypoglycemic sensing in detail. However, recent controversy about the relevance of rodent findings to large animals and humans prompted us to investigate the effect of portal DEN on the hypoglycemic response in the canine, a species with multiple similarities to human glucose homeostasis. Hypoglycemic hyperinsulinemic clamps were performed in male canines, before (PRE) and after (POST) portal vein DEN or sham surgery (CON, control). Insulin (30 pmol/kg·min) and glucose (variable) were infused to slowly decrease systemic glycemia to 50 mg/dL over 160 minutes. The average plasma glucose during clamp steady state was: 2.9 ± 0.1 mmol DEN-PRE, 2.9 ± 0.2 mmol DEN-POST, 2.9 ± 0.1 mmol CON-PRE, and 2.8 ± 0.0 mmol CON-POST. There were no significant differences in plasma insulin between DEN and CON, PRE and POST experiments. The epinephrine response to hypoglycemia was reduced by 62% in DEN but not in CON. Steady-state cortisol was 46% lower after DEN but not after CON. Our study shows, in a large animal model, that surgical disconnection of the portal vein from the afferent pathway of the hypoglycemic counterregulatory circuitry results in a substantial suppression of the epinephrine response and a significant impact on cortisol response. These findings directly demonstrate an essential role for the portal vein in sensing hypoglycemia and relating glycemic information to the central nervous system.

Magnitude and mechanisms of glucose counterregulation following islet transplantation in patients with type 1 diabetes suffering from severe hypoglycaemic episodes

Pancreatic islet transplantation stabilises glycaemic control in type 1 diabetes mellitus patients with neuroglycopoenia, despite them not achieving insulin independence because of limited graft function. However, the extent and underlying metabolic pathways of restored glucose counterregulation are unknown. We therefore compared systemic glucose turnover, including lactate gluconeogenesis (GN) and muscle glucose uptake, in individuals with type 1 diabetes who were transplant recipients with partial graft function (T1DM/ITx(+)), matched non-transplanted individuals with type 1 diabetes (T1DM/ITx(-)) and matched healthy non-diabetic individuals. Participants (n = 12 in each group) underwent a euglycaemic and a hypoglycaemic (2.5-2.8 mmol/l) hyperinsulinaemic clamp (0.8 mU kg(-1) min(-1)) in a randomised crossover fashion. Systemic and skeletal muscle glucose and lactate kinetics were assessed using a combination of isotopic and forearm balance techniques. Whole-body glucose counterregulation, the difference in glucose infusion rates required to maintain the glycaemic goal between the hypoglycaemic and euglycaemic clamps, was improved in T1DM/ITx(+) (7.8 ± 1.3 μmol kg(-1) min(-1)) compared with T1DM/ITx(-) (0.3 ± 0.9 μmol kg(-1) min(-1)), but was ~45% lower than in controls (14.1 ± 2.1 μmol kg(-1) min(-1)). Increased endogenous glucose production (EGP) and decreased systemic glucose disposal accounted for 49% and 39% of glucose counterregulation in T1DM/ITx(+), respectively, compared with 60% and 36% in controls. Lactate GN increased in T1DM/ITx(+) (2.7 ± 0.4 μmol kg(-1) min(-1)) and controls (1.7 ± 0.5 μmol kg(-1) min(-1)), such that it accounted for 70% and 20% of the increased EGP, respectively. Skeletal muscle accounted for similar proportions of the decrease in systemic glucose disposal in controls (49%) and T1DM/ITx(+) (41%). Partial islet graft function improves hypoglycaemia counterregulation by increasing EGP, largely via lactate GN and decreasing systemic glucose disposal. This may explain the reduction in severe hypoglycaemic events in T1DM/ITx(+) individuals. ClinicalTrials.gov NCT01668485.

Lactate and the Mechanism of Hypoglycemia-Associated Autonomic Failure in Diabetes

Iatrogenic hypoglycemia is a problem for many people with diabetes (1). It causes recurrent morbidity in most people with type 1 diabetes and many with advanced type 2 diabetes and is sometimes fatal. It generally precludes maintenance of euglycemia over a lifetime of diabetes and therefore full realization of the benefits of glycemic control. It impairs defenses against subsequent falling plasma glucose concentrations and causes a vicious cycle of recurrent hypoglycemia. Hypoglycemia in diabetes is typically the result of the interplay of therapeutic insulin excess—caused by treatment with insulin, a sulfonylurea, or a glinide—and compromised physiological and behavioral defenses against falling plasma glucose concentrations (1,2). Compromised physiological defenses include loss of the normal decrease in β-cell insulin secretion and increase in α-cell glucagon secretion and attenuation of the normal increase in adrenomedullary epinephrine secretion during hypoglycemia. The compromised behavioral defense is failure to ingest carbohydrates because of loss of symptoms due to attenuation of the normal increase in sympathoadrenal, largely sympathetic neural, activity. The concept of hypoglycemia-associated autonomic failure (HAAF) in diabetes posits that recent antecedent hypoglycemia (or sleep or prior exercise) causes both defective glucose counterregulation (by attenuating the epinephrine response in the setting of absent insulin and glucagon responses) and impaired awareness of hypoglycemia (by attenuating sympathoadrenal and the resulting symptomatic responses) and thus a vicious cycle of recurrent hypoglycemia. Although additional intraislet mechanisms may be involved, it is reasonable to attribute both loss of the insulin and of the glucagon responses to hypoglycemia, the prerequisites to HAAF, to β-cell failure. Insulin normally restrains glucagon secretion and a decrease in insulin normally stimulates glucagon secretion during hypoglycemia. In the setting of absolute endogenous insulin deficiency—β-cell failure—there is …

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

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