Recurrent Insulin-induced Hypoglycemia Research Articles

145-OR: Counterregulatory Response to Hypoglycemia Is Improved with Serotonin and Norepinephrine Reuptake Inhibitors

We previously reported that serotonin and norepinephrine reuptake inhibitors, amitriptyline (AMT) and imipramine (IMP), restored hypoglycemia awareness in rodents. To test the hypothesis that AMT and IMP could prevent the development of an impaired counterregulatory response (CRR) to hypoglycemia, Sprague-Dawley rats (250-350 g) with pre-implanted arterial and jugular cannulae were randomly assigned to one of four treatment groups: 1) recurrent saline controls (RS, n=8); 2) recurrent insulin-induced hypoglycemia (RH, 30-50 mg/dl, n=8); 3) RH + AMT (10 mg/kg IP, n=8); or 4) RH + IMP (5 mg/kg IP, n=8). Following 3-days of RS/RH treatment, all groups underwent 90-minute hyperinsulinemic (50 mU.kg-1.min-1) hypoglycemic (40-45 mg/dL) clamps. Blood glucose was similar for all groups during the clamp. Glucose infusion rates during the last 30-minutes were significantly reduced for both RH+AMT (23.7 mg/kg/min) and RH+IMP (24.8 mg/kg/min) as compared to RH (40.4 mg/kg/min), indicating that both drugs improved the glucoregulatory response to hypoglycemia. In conclusion, these studies identify 1) the involvement of serotonin and norepinephrine neurotransmitter pathways in the development of blunted counterregulatory responses to hypoglycemia, 2) in subjects with blunted CRR, these drugs may be useful therapeutic agents to improve the glucoregulatory response to hypoglycemia. Disclosure E.L.Macon: None. M.H.Devore: None. A.Iles: None. L.A.Schoeder: None. B.Patel: None. M.B.Music: None. S.Fisher: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK118082)

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

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

231-OR: Antecedent Hypoglycemia Increases Damage Due to a Subsequent Myocardial Infarction in Rats

Several clinical trials have noted an association between hypoglycemia and major cardiovascular events; however, a direct link between antecedent hypoglycemia and worse cardiac outcomes has not been established. We hypothesized that antecedent recurrent hypoglycemia predisposes the heart to increased damage following a subsequent myocardial infarction. To test this hypothesis, adult Sprague Dawley rats were subjected to 3 days of recurrent insulin-induced hypoglycemia (25-30 mg/dl, 90 minutes each day; n = 6) or saline (control, n = 6) . On the following day, all rats underwent a myocardial infarction (MI) induced by occlusion of the left anterior descending artery for 30 minutes followed by reperfusion for 60 minutes. Electrocardiograms indicated ST elevation during the MI, which returned to normal sinus rhythm upon reperfusion. Hearts were removed immediately and analyzed for damage. MI induced ventricular damage from rats that underwent antecedent recurrent hypoglycemia (39 ± 5%) was significantly higher than control rats (16 ± 4%; p<0.001, Figure) . Additionally, plasma norepinephrine levels were elevated 14-fold in rats that underwent recurrent hypoglycemia. These studies indicate that antecedent hypoglycemia directly contributes to worse outcomes following an MI and that increased norepinephrine is associated with this damage. Disclosure C.G.Christensen: None. S.Fisher: None. C.M.Reno: None. Funding University of Utah’s Driving Out Diabetes, a Larry H. Miller Family Wellness InitiativeNational Institutes of Health (P30 DK020579) Juvenile Diabetes Research Foundation (1-FAC-2020-984-A-N)

Hindbrain catecholamine regulation of ventromedial hypothalamic nucleus glycogen metabolism during acute versus recurring insulin-induced hypoglycemia in male versus female rat

Ventromedial hypothalamic nucleus (VMN) glycogen metabolism affects local glucoregulatory signaling. The hindbrain metabolic-sensitive catecholamine (CA) neurotransmitter norepinephrine controls VMN glycogen phosphorylase (GP)-muscle (GPmm) and -brain (GPbb) type expression in male rats. Present studies addressed the premise that CA regulation of hypoglycemic patterns of VMN glycogen metabolic enzyme protein expression is sex-dimorphic, and that this signal is responsible for sex differences in acclimation of these profiles to recurrent insulin-induced hypoglycemia (RIIH). VMN tissue was acquired by micropunch-dissection from male and female rats pretreated by caudal fourth ventricular administration of the CA neurotoxin 6-hydroxydopamine (6OHDA) before single or serial insulin injection. 6-OHDA averted acute hypoglycemic inhibition of VMN glycogen synthase (GS) and augmentation of GPmm and GPbb protein expression in males, and prevented GPmm and -bb down-regulation in females. Males recovered from antecedent hypoglycemia (AH) exhibited neurotoxin-preventable diminution of baseline GS profiles, whereas acclimated GPmm and -bb expression in females occurred irrespective of pretreatment. RIIH did not alter VMN GS, GPmm, and GPbb expression in vehicle- or 6-OHDA-pretreated animals of either sex. VMN glycogen content was correspondingly unchanged or increased in males versus females following AH; 6-OHDA augmented glycogen mass in AH-exposed animals of both sexes. RIIH did not alter VMN glycogen accumulation in vehicle-pretreated rats of either sex, but diminished glycogen in neurotoxin-pretreated animals. AH suppresses baseline GS (CA-dependent) or GPmm/GPbb (CA-independent) expression in male and female rats, respectively, which corresponds with unaltered or augmented VMN glycogen content in those sexes. AH-associated loss of sex-distinctive CA-mediated enzyme protein sensitivity to hypoglycemia (male: GS, GPmm, GPbb; female: GPmm, Gpbb) may reflect, in part, VMN target desensitization to noradrenergic input.

Sex-specific acclimation of A2 noradrenergic neuron dopamine-β-hydroxylase and estrogen receptor variant protein and 5’-AMP-Activated protein kinase reactivity to recurring hypoglycemia in rat

Hindbrain estrogen receptors (ER) impose sex-dimorphic control of counter-regulatory hormone and hypothalamic glucoregulatory transmitter and glycogen metabolic responses to hypoglycemia. A2 noradrenergic neurons are estradiol- and metabolic-sensitive. Estradiol controls dopamine-beta-hydroxylase (DBH) protein habituation to recurrent insulin-induced hypoglycemia (RIIH) in females. Current research investigated the premise that sex-dimorphic patterns of A2 ER variant acclimation to RIIH correlate with differential A2 DBH and 5’-AMP-activated protein kinase (AMPK) adaptation to RIIH. A2 neurons were laser-catapult-microdissected from male and female rats after one or four insulin injections for Western blot analysis. A2 pAMPK and DBH levels were increased in males, but suppressed in females after single insulin dosing. ER-alpha (ERα) and -beta (ERβ) protein profiles were unaffected or decreased by acute hypoglycemia in each sex, whereas G protein-linked ER-1 (GPER) reactivity varied by sex. Antecedent hypoglycemia diminished basal A2 ERα/GPER and elevated ERβ content in each sex, yet reduced pAMPK and DBH levels in female rats only. Reintroduced hypoglycemia suppressed A2 ERβ levels in each sex, but altered DBH (↓), ERα (↓), and GPER (↑) levels in males only. Data document sex differences in A2 DBH adaptation to RIIH, e.g. a shift from positive-to-negative response in males versus loss of negative reactivity in females, as well as attenuated AMPK activation in both sexes. Between hypoglycemic episodes, A2 neurons in each sex likely exhibit diminished sensitivity to ERα/GPER signaling, but heightened receptivity to ERβ input. RIIH-induced changes in ERα and GPER expression in male but not female may contribute to DBH suppression (males) versus no change (females) relative to adapted baseline expression.

Effects of acute versus recurrent insulin-induced hypoglycemia on ventromedial hypothalamic nucleus metabolic-sensory neuron AMPK activity: Impact of alpha1-adrenergic receptor signaling

Mechanisms that underlie metabolic sensor acclimation to recurring insulin-induced hypoglycemia (RIIH) are unclear. Norepinephrine (NE) regulates ventromedial hypothalamic nucleus (VMN) gluco-stimulatory nitric oxide (NO) and gluco-inhibitory γ-aminobutryic acid (GABA) neuron signaling. Current research addressed the hypothesis that during RIIH, NE suppresses 5′-AMP-activated protein kinase (AMPK) reactivity in both populations and impedes counter-regulation. The brain is postulated to utilize non-glucose substrates, e.g. amino acids glutamine (Gln), glutamate (Glu), and aspartate (Asp), to produce energy during hypoglycemia. A correlated aim investigated whether NE controls pyruvate recycling pathway marker protein (glutaminase, GLT; malic enzyme, ME-1) expression in either metabolic-sensory cell population. Male rats were injected subcutaneously with vehicle or insulin on days 1–3, then pretreated on day 4 by intracerebroventricular delivery of the alpha1-adrenergic receptor (α1-AR) reverse-agonist prazocin (PRZ) or vehicle before final insulin therapy. PRZ prevented acute hypoglycemic augmentation of AMPK activation in each cell group. Antecedent hypoglycemic repression of sensor activity was reversed by PRZ in GABA neurons. During RIIH, nitrergic neurons exhibited α1-AR – dependent up-regulated GLT and α2-AR profiles, while GABA cells showed down-regulated α1-AR. LC-ESI-MS analysis documented a decline in VMN Glu, Gln, and Asp concentrations during acute hypoglycemia, and habituation of the former two profiles to RIIH. PRZ attenuated glucagon and corticosterone secretion during acute hypoglycemia, but reversed decrements in output of both hormones during RIIH. Results implicate adjustments in impact of α1-AR signaling in repressed VMN metabolic-sensory AMPK activation and counter-regulatory dysfunction during RIIH. Antecedent hypoglycemia may up-regulate NO neuron energy yield via α1-AR – mediated up-regulated pyruvate recycling.

MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia.

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 GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3'-UTR of GABAA 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.

Recurrent insulin-induced hypoglycemia induces AngII and COX2 leading to renal (pro)renin receptor expression and oxidative stress

Background: Recurrent insulin-induced hypoglycemia (RIIH) is an avoidable consequence in the therapeutic management of diabetes mellitus. RIIH has been implicated in causing hypertension through an increase in renal and systemic AngII production.Objective: The present study was performed to assess the hypothesis that chronic insulin treatment enhances AngII and COX2 formation which in turn increases (pro) renin receptor (PRR) expression and NADPH oxidase-mediated oxidative stress, leading to renal and cardiac injury.Methods: The present studies were conducted in Male Sprague Dawley rats treated with daily subcutaneous injections of 7u/kg insulin or saline for 14 days. On the 14th day, surgery was performed for treatment infusion (captopril 12mg/kg, NS398 0.3mg/kg or vehicle), and renal interstitial fluid sample and urine collections for biomarker measurements. At the end of the experiments, kidneys and hearts were harvested to evaluate PRR and NOX2 (NADPH oxidase subunit) expression and oxidative stress.Results: We found that RIIH enhanced AngII and COX2 activity, leading to renal PRR expression and NADPH oxidase-induced oxidative stress in the heart and kidney. 8-isoprostane was evaluated as a renal biomarker of oxidative stress, which was induced in insulin treated animals and modulated by captopril and NS398. In addition, there was a slight increase in NGAL, a urinary biomarker of acute kidney injury (AKI), in insulin treated animals when compared to control.Conclusion: These results demonstrate that RIIH induces renal PRR expression and oxidative stress through increasing AngII and COX2 in the heart and kidney, leading to end-organ damage.

Evaluating the significance of hypoglycemia in promoting insulin induced hypertension by using a glucose clamp model

Background: Hypoglycemia unawareness is a detrimental risk factor in diabetic patients leading to serious complications if left untreated. Previous studies from our group determined that recurrent insulin induced hypoglycemia (RIIH) promotes hypertension.Objective: We hypothesize that RIIH not insulin by itself is the source of hypertension and end organ damage in diabetic patients.Methods: Male Sprague-Dawley-rats (200-250g, n=18) were provided with glucose food (125g glucose/kg body weight) and glucose water (0.1g glucose/100g body weight/ml). They were treated with subcutaneous insulin injections (7U/Kg) and blood glucose was monitored intermittently. Daily blood pressure was measured using the tail cuff method. Interstitial samples of ATP and angiotensinII (AngII) were collected by renal microdialysis and analyzed using luciferin-luciferase bioluminescent assay and EIA respectively. Reactive oxygen and nitrogen species in hearts and kidneys were analyzed using Electron Paramagnetic Resonance Spectroscopy (EPR).Results: Renal interstitial ATP levels increased from 90.2± 4.7ng/µl to 99.6± 8.7ng/µl (not significant) and AngII from 0.15± 0.02ng/ml to 0.13± 0.05ng/ml (not significant) from day 0 to 14. There was no significant change in mean arterial pressure (121.3 ± 1.4 mmHg on day 0 to 127.8 ± 1.4mmHg on day 14). Oxidative stress was reduced compared to RIIH model, which was evident from the EPR spectra.Conclusion: We demonstrated that hypertension induced end organ damage in diabetics is due to insulin induced hypoglycemia not insulin alone (by itself).

Impact of recurrent hypoglycemic stress on hindbrain A2 nerve cell energy metabolism and catecholamine biosynthesis: modulation by estradiol.

It is unclear if habituation of hindbrain A2 metabolo‑sensory neurons to recurrent insulin-induced hypoglycemia (RIIH) correlates with estradiol-dependent adjustments in energy metabolism that favor positive energy balance. Laser-microdissected A2 cells from estradiolor oil-implanted ovariectomized female rats were analyzed by Western blot to assess effects of three prior daily insulin injections on basal and hypoglycemic patterns of catecholamine biosynthetic enzyme dopamine-beta-hydroxylase (DβH) and rate-limiting energy pathway enzyme protein expression. Precedent hypoglycemia respectively decreased or increased baseline DβH expression in estradiol- (E) vs. oil (O)-treated rats; this protein profile was further suppressed or augmented in those animals at 2 hr after re-induction of hypoglycemia. These data suggest that estradiol may curtail A2 noradrenergic‑controlled functions both in the midst of and between hypoglycemic bouts. Results also show that prior hypoglycemia exposure upregulated A2 neuron glycolytic enzyme protein levels when E was present, and exerted differential effects on basal and hypoglycemia-associated respiratory chain and fatty acid synthetic pathway enzyme expression. E may thus accordingly amplify glycolysis-derived metabolites/energy, coupled with reduced reliance on oxidative phosphorylation, and activate the fatty acid synthetic pathway during RIIH. E may also be of benefit by preventing maladaptive reductions in A2 neuron Krebs cycle/electron transport enzyme expression during re-exposure to hypoglycemia. Augmentation of negative energy balance during this recurring metabolic stress in the absence of E is a likely impetus for augmented vs. decreased A2 signaling of energy imbalance by DβH in O vs. E rats during RIIH.

The Role of Recurrent Insulin-Induced Hypoglycemia on Renal Prostanoid Production

The Role of Recurrent Insulin-Induced Hypoglycemia on Renal Prostanoid Production

Orexin signaling is necessary for hypoglycemia-induced prevention of conditioned place preference.

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Role of Interstitial Angiotensin II and ATP in Mediating Renal Injury Induced by Recurrent Insulin Induced Hypoglycemia

Aim: The present study hypothesizes that recurrent insulin induced hypoglycemia (RIIH) elevates renal interstitial ATP levels which in turn enhances AngII production. This interrupts the normal tubuloglomerular feedback (TGF) mechanism by stimulating afferent arteriolar vasoconstriction resulting in hypertension, which augments oxidative stress and could promote renal damage. Study Design: In the present study we adopted a microdialysis technique, which is a minimally invasive tool for monitoring chronic changes in renal interstitial fluid. Place and Duration of Study: Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe between September 2012 – October 2013. Methodology: Eight male Sprague Dawley rats (200-225 g) were anesthetized and microdialysis probes were inserted into their renal cortex. Post-surgery rats were treated with insulin (7U/kg body Original Research Article Prathipati et al.; ARRB, 6(5): 328-336, 2015; Article no.ARRB.2015.092 329 weight) for 2 weeks. Food and water intake were monitored daily. Physiological saline was perfused through the probe and dialysate was collected daily after insulin dosing and analyzed for ATP by luciferin-luciferase assay and AngII by EIA. At the end of the experiment, the hearts and kidneys were collected and analyzed for oxidative stress by EPR (Electron Paramagnetic Resonance) spectroscopy using CMH and CPH spin probes. Results: ATP and AngII levels were elevated from 31.65±4.4ng/μl (day 0) to 130.96±2.9 ng/μl (day 14) and 0.1±0.01 ng/ml (day 0) to 0.247±0.02 ng/ml(day 14), respectively. Elevation of peroxynitrite and superoxide anions were observed in the hearts and kidneys of insulin treated animals when compared to the control group. Conclusion: Thus the present study utilizes real-time chronic collections of renal interstitial samples to identify a potential mechanism where iatrogenic hypoglycemia promotes hypertension via a synergistic relationship between interstitial ATP, AngII and developed oxidative stress.

Regulation of heme oxygenase-1 induction during recurrent insulin induced hypoglycemia

Background: Recurrent insulin induced hypoglycemia (RIIH) is an unavoidable risk of conventional therapeutic management of insulin dependent diabetes mellitus and is a primary cause of impaired glucose counter regulation. Circulating angiotensin II (AngII) has been reported to be elevated in diabetic models, and is linked to the promotion of hypertension. Objective: The current study was performed to evaluate the hypothesis that chronic insulin injections and/or hypoglycemia promotes hypertension via an increase in AngII, which increases endogenous carbon monoxide (CO) through an induction in heme oxygenase (HO-1). Methods: Male Sprague Dawley rats (200 250g) were treated for 2 weeks with daily injections of 7U/kg insulin or diluent. On the 14th day, surgery was performed and rats were administered various doses of captopril (2mg/kg, 8mg/kg, and 12mg/kg) or vehicle. Following the dose evaluation, subsets of previously treated animals were treated with vehicle, DALA (80g/kg), Captopril (12mg/kg) or a combination of DALA (80g/kg) + CAP (12mg/kg). Results: A dose dependent decrease in blood pressure was observed during captopril treatment in RIIH hypertensive rats. However, there was no change in urine output among the treatment groups. Captopril and DALA+CAP treatments produced a reduction in blood pressure as compared to animals treated with DALA alone. Carboxyhemoglobin and AngII concentrations were also reduced in animals treated with captopril and DALA+CAP. In addition, HO-1 protein levels in heart and kidney were reduced when compared to DALA treated animals. Conclusion: These results demonstrate that RIIH promotes an increase in circulating AngII and hypertension via an induction in HO-1, which significantly increases endogenous CO levels. Keywords: Angiotensin II, Carbon Monoxide, Heme Oxygenase, Recurring Insulin-Induced Hypoglycemia (RIIH).

Site-Specific Effects of Intracranial Estradiol Administration on Recurrent Insulin-Induced Hypoglycemia in the Ovariectomized Female Rat

Clinical and experimental studies reveal gender differences in susceptibility to dampening effects of precedent hypoglycemia on recurrent insulin-induced hypoglycemia (RIIH). Recent studies implicate the ovarian steroid, estradiol, in the regulation of RIIH, since systemic replacement of this hormone at basal estrous cycle levels maintains glucose profiles during serial insulin dosing and prevents RIIH-associated reductions in neuronal activation in key metabolic structures in the ovariectomized female rat brain. The present study investigated the hypothesis that these effects are achieved, in part, by estrogenic action within the central nervous system, including glucoregulatory structures characterized by high estrogen receptor (ER) expression. Initial experiments evaluated the impact of global intracranial administration of estradiol on RIIH. Ovariectomized rats were treated by continuous infusion of graded doses of 17β-estradiol-3-benzoate (EB) or vehicle into the lateral ventricle (LV), and injected subcutaneously with 1 or 4 doses of the intermediate-release insulin, Humulin N (HN), 1 dose per day. Animals infused with 5 or 10 µg EB/day exhibited uniform glycemic responses to 1 versus 4 doses of insulin, whereas rescue from hypoglycemia was delayed during repetitive HN injection of rats infused with either vehicle or 1 µg EB/day. Recovery from both single and multiple bouts of hypoglycemia was more rapid in rats infused with the higher EB doses, compared to other groups. Mapping of ERα immunoreactivity in animals treated by LV infusion of EB revealed variable nuclear staining in ER-expressing metabolic loci typified by estrogen-dependent sustenance of neuronal reactivity to hypoglycemia, with highest levels of ERα immunoreactivity observed in the arcuate (ARH) and ventromedial (VMH) hypothalamic nuclei, and moderate labeling of the caudal hindbrain dorsal vagal complex. EB delivery to the caudal hindbrain via the caudal fourth ventricle resulted in dose-dependent effects on RIIH, since glycemic profiles were either unchanged or diminished relative to acute NH-induced hypoglycemia, in high versus low EB-treated animals, respectively. Bilateral administration of 1.0 µg EB into the ARH or VMH elicited disparate effects on acute and chronic HN-induced hypoglycemia. Intra-VMH EB delayed recovery from both acute and chronic hypoglycemia, compared to non-estradiol-treated controls. In contrast, neither that dose nor a 10-fold lower dosage of EB delivered to the ARH modified acute HN-induced hypoglycemia, but RIIH was either attenuated or enhanced, respectively, in animals treated by intra-ARH delivery of 1.0 versus 0.1 µg EB, respectively. These results suggest that whole brain exposure to elevated estradiol may promote outflow that truncates hypoglycemia and maintains glucose profiles during RIIH, whereas actions of relatively low hormone levels on the central nervous system may result in adaptive adjustments that result in lower blood glucose levels during recurring versus acute hypoglycemia. The data also imply that, at a given concentration, estrogens may exert site-specific effects on acute and chronic HN-induced hypoglycemia. Further research is needed to identify the cellular substrates and physiological mechanisms that mediate caudal hindbrain-, ARH-, and VMH-specific actions of estradiol on acute and chronic hypoglycemia.

Recurrent hypoglycemia increases hypothalamic glucose phosphorylation activity in rats

The mechanisms underpinning impaired defensive counterregulatory responses to hypoglycemia that develop in some people with diabetes who suffer recurrent episodes of hypoglycemia are unknown. Previous work examining whether this is a consequence of increased glucose delivery to the hypothalamus, postulated to be the major hypoglycemia-sensing region, has been inconclusive. Here, we hypothesized instead that increased hypothalamic glucose phosphorylation, the first committed intracellular step in glucose metabolism, might develop following exposure to hypoglycemia. We anticipated that this adaptation might tend to preserve glucose flux during hypoglycemia, thus reducing detection of a falling glucose. We first validated a model of recurrent hypoglycemia in chronically catheterized (right jugular vein) rats receiving daily injections of insulin. We confirmed that this model of recurrent insulin-induced hypoglycemia results in impaired counterregulation, with responses of the key counterregulatory hormone, epinephrine, being suppressed significantly and progressively from the first day to the fourth day of insulin-induced hypoglycemia. In another cohort, we investigated the changes in brain glucose phosphorylation activity over 4 days of recurrent insulin-induced hypoglycemia. In keeping with our hypothesis, we found that recurrent hypoglycemia markedly and significantly increased hypothalamic glucose phosphorylation activity in a day-dependent fashion, with day 4 values 2.8 ± 0.6-fold higher than day 1 ( P < .05), whereas there was no change in glucose phosphorylation activity in brain stem and frontal cortex. These findings suggest that the hypothalamus may adapt to recurrent hypoglycemia by increasing glucose phosphorylation; and we speculate that this metabolic adaptation may contribute, at least partly, to hypoglycemia-induced counterregulatory failure.

Adaptation of Feeding and Counter‐Regulatory Hormone Responses to Intermediate Insulin‐Induced Hypoglycaemia in the Ovariectomised Female Rat: Effects of Oestradiol

Oestradiol regulates basal food intake and glucagon and corticosterone secretion, but its influence on these responses to acute and recurring hypoglycaemia remains unclear. The present study utilised an experimental model for repeated intermediate-acting insulin-induced hypoglycaemia that replicates the route of delivery, frequency of administration, and duration of insulin action in the clinical setting. Groups of ovariectomised (OVX) rats were implanted with s.c. capsules containing oestradiol benzoate (EB) or oil, and injected with one or four doses of Humulin neutral protamine Hagedorn (HN), on as many days, or diluent alone. Baseline feeding followed divergent trends in EB- versus oil-implanted animals over a 9-h period after final injections. Recurring HN-induced hypoglycaemia resulted in significantly greater baseline-corrected food intake in OVX + EB and OVX + oil groups, relative to acute hypoglycaemic hyperphagia. Although oestradiol did not modify net food consumption after single or serial HN doses, EB replacement maintained uniform feeding over time in each treatment paradigm. Baseline glucagon and corticosterone secretion was higher in EB- versus oil-treated OVX rats. Oestradiol prolonged acute hypoglycaemic glucagonemia, and increased the magnitude, but shortened the duration, of glucagon secretion during recurring hypoglycaemia. OVX + oil rats responded to both acute and recurring hypoglycaemia with elevated corticosterone secretion at a single time point, which was advanced from +6 to +4 h during recurrent insulin-induced hypoglycemia, whereas OVX + EB animals exhibited increased plasma hormone levels at both +4 and +6 h in response to each paradigm. Area-under-the curve analyses showed that total glucagon and corticosterone release was greater in EB- versus oil-implanted rats after both single and serial dosing with HN. These results demonstrate that repeated HN administration increases food intake in female rats via oestrogen-independent mechanisms, but that oestradiol preserves temporal patterns of hypoglycaemic hyperphagia. The data also reveal that normo- and hypoglyacemic glucagon and corticosterone secretion are enhanced in the presence of oestrogen. Further studies are necessary to identify the sites and cellular substrates that are responsible for this hormonal regulation of behavioural and endocrine responses to prolonged hypoglycaemia.

Recurrent Antecedent Hypoglycemia Alters Neuronal Oxidative Metabolism In Vivo

OBJECTIVEThe objective of this study was to characterize the changes in brain metabolism caused by antecedent recurrent hypoglycemia under euglycemic and hypoglycemic conditions in a rat model and to test the hypothesis that recurrent hypoglycemia changes the brain's capacity to utilize different energy substrates.RESEARCH DESIGN AND METHODSRats exposed to recurrent insulin-induced hypoglycemia for 3 days (3dRH rats) and untreated controls were subject to the following protocols: [2-13C]acetate infusion under euglycemic conditions (n = 8), [1-13C]glucose and unlabeled acetate coinfusion under euglycemic conditions (n = 8), and [2-13C]acetate infusion during a hyperinsulinemic-hypoglycemic clamp (n = 8). In vivo nuclear magnetic resonance spectroscopy was used to monitor the rise of13C-labeling in brain metabolites for the calculation of brain metabolic fluxes using a neuron-astrocyte model.RESULTSAt euglycemia, antecedent recurrent hypoglycemia increased whole-brain glucose metabolism by 43 ± 4% (P < 0.01 vs. controls), largely due to higher glucose utilization in neurons. Although acetate metabolism remained the same, control and 3dRH animals showed a distinctly different response to acute hypoglycemia: controls decreased pyruvate dehydrogenase (PDH) flux in astrocytes by 64 ± 20% (P = 0.01), whereas it increased by 37 ± 3% in neurons (P = 0.01). The 3dRH animals decreased PDH flux in both compartments (−75 ± 20% in astrocytes, P < 0.001, and −36 ± 4% in neurons, P = 0.005). Thus, acute hypoglycemia reduced total brain tricarboxylic acid cycle activity in 3dRH animals (−37 ± 4%, P = 0.001), but not in controls.CONCLUSIONSOur findings suggest that after antecedent hypoglycemia, glucose utilization is increased at euglycemia and decreased after acute hypoglycemia, which was not the case in controls. These findings may help to identify better methods of preserving brain function and reducing injury during acute hypoglycemia.

Role of dorsal vagal motor nucleus orexin-receptor-1 in glycemic responses to acute versus repeated insulin administration

The potent orexigenic neuropeptide, orexin-A (ORX-A), acts at multiple sites within the central neuroaxis to control autonomic responses to energy imbalance, including the dorsal vagal motor nucleus (DMV), where it regulates pancreatic efferent nerve firing. Recent evidence that recurrent insulin-induced hypoglycemia (RIIH) attenuates lateral hypothalamic ORX-A-ergic neuronal transcriptional activation and prepro-orexin gene expression suggests that this phenotype undergoes functional adaptation to repeated glucoprivation. We examined the hypothesis that RIIH-associated patterns of ORX-A neurotransmission and/or orexin-receptor-1 (OR-1) expression within the DMV may be correlated with exacerbated hypoglycemic and impaired pancreatic counterregulatory responses to repeated insulin administration. Male rats were pretreated by bilateral intra-DMV infusion of the OR-1 antagonist, SB-334867, or vehicle prior to s.c. injection of Humulin NPH (NPH), or diluent alone. Other animals were injected with one or four doses of NPH, on as many days, or diluent alone, and pretreated by bilateral intra-DMV administration of graded doses of ORX-A or vehicle on the final day of the study. Effects of acute versus repeated insulin administration on ORX-A and OR-1 protein levels in the microdissected dorsal vagal complex (DVC) were evaluated by radioimmunoassay and Western blot analyses, respectively. SB-334867 treatment prior to acute NPH administration decreased plasma glucose and suppressed peak glucagon secretion, whereas exogenous ORX-A administration prior to RIIH did not reverse amplified patterns of hypoglycemia. RIIH did not alter intra-DVC ORX-A tissue concentrations, but diminished OR-1 levels in that site. These results show that DMV OR-1 function is critical for optimal glucagon secretory responsiveness to acute hypoglycemia, and that RIIH-associated downregulation of receptor expression in that brain site may contribute to impaired restoration of euglycemia. The current data provide unique evidence that ORX-A acts via OR-1-dependent mechanisms within DMV to regulate glucagon counterregulatory function during hypoglycemia, and that decreased receptor-mediated signaling during RIIH may underlie characteristic intensification of hypoglycemia.

Effects of Estradiol on Glycemic and CNS Neuronal Activational Responses to Recurrent Insulin-Induced Hypoglycemia in the Ovariectomized Female Rat

Recurrent insulin-induced hypoglycemia (RIIH) results in glucose counterregulatory dysfunction in men and male rodents. Intensified hypoglycemia in the latter coincides with diminished neuronal Fos expression in central metabolic regulatory structures, evidence that supports habituation of CNS-mediated compensatory motor outflow during re-exposure to this metabolic stress. In light of the evidence for counterregulatory resistance to precedent hypoglycemia in women, we utilized estradiol-treated ovariectomized (OVX) female rats to examine the hypothesis that this hormone regulates neural adaptability to recurring hypoglycemia. Groups of OVX rats were implanted with subcutaneous silastic capsules containing estradiol benzoate (E) or oil alone, and injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, one dose daily, or with diluent alone. Blood glucose levels were not altered by RIIH in E-implanted OVX animals, but were significantly decreased after four versus one insulin injection in the OVX+oil group. Mean numbers of Fos-immunoreactive (ir) neurons in the paraventricular nucleus hypothalamus (PVH), dorsomedial nucleus hypothalamus (DMH), and lateral hypothalamic area (LHA) were higher in both E- versus oil-implanted OVX rats injected with diluent only. Acute hypoglycemia significantly increased mean counts of Fos-ir-positive neurons in the PVH, DMH, and LHA, as well as the nucleus of the solitary tract (NTS) and area postrema (AP) in E- and oil-treated animals to an equivalent extent. OVX+E rats exhibited comparable numbers of Fos-positive neurons in the PVH, DMH, and LHA after one versus four insulin injections, whereas the numbers of labeled neurons in NTS and AP were increased or decreased, respectively, by RIIH. Oil-implanted OVX rats showed significantly diminished numbers of Fos-ir-positive neurons in each neural structure after repeated hypoglycemia. The present data demonstrate that estradiol sustains or enhances neuronal reactivity to recurring hypoglycemia in central metabolic structures, whereas hypoglycemic patterns of Fos expression in each site become habituated during RIIH in the absence of this steroid. The brain sites characterized here by estrogen-dependent maintenance of neuronal genomic reactivity to this substrate fuel imbalance may contain direct and/or indirect cellular targets for hormonal actions that prevent adaptation of CNS-controlled motor responses to this metabolic stress.