Counterregulatory Hormone Secretion Research Articles

Disrupted hypothalamic transcriptomics and proteomics in a mouse model of type 2 diabetes exposed to recurrent hypoglycaemia

Aims/hypothesisRepeated exposures to insulin-induced hypoglycaemia in people with diabetes progressively impairs the counterregulatory response (CRR) that restores normoglycaemia. This defect is characterised by reduced secretion of glucagon and other counterregulatory hormones. Evidence indicates that glucose-responsive neurons located in the hypothalamus orchestrate the CRR. Here, we aimed to identify the changes in hypothalamic gene and protein expression that underlie impaired CRR in a mouse model of defective CRR.MethodsHigh-fat-diet fed and low-dose streptozocin-treated C57BL/6N mice were exposed to one (acute hypoglycaemia [AH]) or multiple (recurrent hypoglycaemia [RH]) insulin-induced hypoglycaemic episodes and plasma glucagon levels were measured. Single-nuclei RNA-seq (snRNA-seq) data were obtained from the hypothalamus and cortex of mice exposed to AH and RH. Proteomic data were obtained from hypothalamic synaptosomal fractions.ResultsThe final insulin injection resulted in similar plasma glucose levels in the RH group and AH groups, but glucagon secretion was significantly lower in the RH group (AH: 94.5±9.2 ng/l [n=33]; RH: 59.0±4.8 ng/l [n=37]; p<0.001). Analysis of snRNA-seq data revealed similar proportions of hypothalamic cell subpopulations in the AH- and RH-exposed mice. Changes in transcriptional profiles were found in all cell types analysed. In neurons from RH-exposed mice, we observed a significant decrease in expression of Avp, Pmch and Pcsk1n, and the most overexpressed gene was Kcnq1ot1, as compared with AH-exposed mice. Gene ontology analysis of differentially expressed genes (DEGs) indicated a coordinated decrease in many oxidative phosphorylation genes and reduced expression of vacuolar H+- and Na+/K+-ATPases; these observations were in large part confirmed in the proteomic analysis of synaptosomal fractions. Compared with AH-exposed mice, oligodendrocytes from RH-exposed mice had major changes in gene expression that suggested reduced myelin formation. In astrocytes from RH-exposed mice, DEGs indicated reduced capacity for neurotransmitters scavenging in tripartite synapses as compared with astrocytes from AH-exposed mice. In addition, in neurons and astrocytes, multiple changes in gene expression suggested increased amyloid beta (Aβ) production and stability. The snRNA-seq analysis of the cortex showed that the adaptation to RH involved different biological processes from those seen in the hypothalamus.Conclusions/interpretationThe present study provides a model of defective counterregulation in a mouse model of type 2 diabetes. It shows that repeated hypoglycaemic episodes induce multiple defects affecting all hypothalamic cell types and their interactions, indicative of impaired neuronal network signalling and dysegulated hypoglycaemia sensing, and displaying features of neurodegenerative diseases. It also shows that repeated hypoglycaemia leads to specific molecular adaptation in the hypothalamus when compared with the cortex.Data availabilityThe transcriptomic dataset is available via the GEO (http://www.ncbi.nlm.nih.gov/geo/), using the accession no. GSE226277. The proteomic dataset is available via the ProteomeXchange data repository (http://www.proteomexchange.org), using the accession no. PXD040183.Graphical

4-LB: Norepinephrine-Stimulated Glutamate Release in the Ventromedial Hypothalamus Is Important to Enhance the Counterregulatory Response during Hypoglycemia

We previously showed that norepinephrine acts through β2-adrenergic receptors to stimulate the release of glutamate in the ventromedial hypothalamus (VMH) and the secretion of counter-regulatory hormones under basal non-stimulated conditions. The current study investigates the importance of this interaction during hypoglycemia and more specifically, whether norepinephrine is an absolute requirement for glutamate secretion in the VMH in response to hypoglycemia and/or whether glutamate neurons respond to hypoglycemia independent of this input. To address this question, we locally delivered the β2-adrenergic receptor blocker, ICI-118,551, into the VMH and evaluated glutamate release in the VMH using microdialysis, as well as its effects on the counter-regulatory hormone responses during a hypoglycemic clamp. These responses were compared to control animals given artificial extracellular fluid. Our data shows that β2-adrenergic receptor blockade reduced VMH glutamate levels, and this was accompanied by a 38% reduction in plasma epinephrine secretion during hypoglycemia (P&amp;lt;0.05 vs Control). Our data suggest that during hypoglycemia, noradrenergic inputs may help drive the release of neurotransmitter glutamate in the VMH that in turn, enhances the release of counter-regulatory hormones. Disclosure I. E. Nelson: None. D. A. Appadurai: None. C. Uzo: None. O. Chan: None.

Sex-Dimorphic Octadecaneuropeptide (ODN) Regulation of Ventromedial Hypothalamic Nucleus Glucoregulatory Neuron Function and Counterregulatory Hormone Secretion.

Central endozepinergic signaling is implicated in glucose homeostasis. Ventromedial hypothalamic nucleus (VMN) metabolic monitoring governs glucose counter-regulation. VMN glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) neurons express the energy gauge 5'-AMP-activated protein kinase (AMPK). Current research addresses the premise that the astrocyte glio-peptide octadecaneuropeptide (ODN) imposes sex-dimorphic control of metabolic sensor activity and neurotransmitter signaling in these neurons. The ODN G-protein coupled-receptor antagonist cyclo(1-8)[DLeu5]OP (LV-1075) was administered intracerebroventricularly (icv) to euglycemic rats of each sex; additional groups were pretreated icv with the ODN isoactive surrogate ODN11-18 (OP) before insulin-induced hypoglycemia. Western blotting of laser-catapult-microdissected VMN NO and GABA neurons showed that hypoglycemia caused OP-reversible augmentation of phospho-, e.g., activated AMPK and nitric oxide synthase (nNOS) expression in rostral (female) or middle (male) VMN segments or ODN-dependent suppression of nNOS in male caudal VMN. OP prevented hypoglycemic down-regulation of glutamate decarboxylase profiles in female rat rostral VMN, without affecting AMPK activity. LV-1075 treatment of male, not female rats elevated plasma glucagon and corticosterone concentrations. Moreover, OP attenuated hypoglycemia-associated augmentation of these hormones in males only. Results identify, for each sex, regional VMN metabolic transmitter signals that are subject to endozepinergic regulation. Directional shifts and gain-or-loss of ODN control during eu- versus hypoglycemia infer that VMN neuron receptivity to or post-receptor processing of this stimulus may be modulated by energy state. In male, counter-regulatory hormone secretion may be governed principally by ODN-sensitive neural pathways, whereas this endocrine outflow may be controlled by parallel, redundant ODN-dependent and -independent mechanisms in female.

Pancreas Transplantation: Current Challenges, Considerations, and Controversies.

Pancreas transplantation (PTx) reestablishes an autoregulating source of endogenous insulin responsive to normal feedback controls. In addition to achieving complete β-cell replacement that frees the patient with diabetes from the need to monitor serum glucose and administer exogenous insulin, successful PTx provides counterregulatory hormone secretion and exocrine function. A functioning PTx mitigates glycemic variability, eliminates the daily stigma and burden of diabetes, restores normal glucose homeostasis in patients with complicated diabetes, and improves quality of life and life expectancy. The tradeoff is that it entails a major surgical procedure and requisite long-term immunosuppression. Despite the high likelihood of rendering patients euglycemic independent of exogenous insulin, PTx is considered a treatment rather than a cure. In spite of steadily improving outcomes in each successive era coupled with expansion of recipient selection criteria to include patients with a type 2 diabetes phenotype, a decline in PTx activity has occurred in the new millennium related to a number of factors including: (1) lack of a primary referral source and general acceptance by the diabetes care community; (2) absence of consensus criteria; and (3) access, education, and resource issues within the transplant community. In the author's experience, patients who present as potential candidates for PTx have felt as though they needed to circumvent the conventional diabetes care model to gain access to transplant options. PTx should be featured more prominently in the management algorithms for patients with insulin requiring diabetes who are failing exogenous insulin therapy or experiencing progressive diabetic complications regardless of diabetes type. Furthermore, all patients with diabetes and chronic kidney disease should undergo consideration for simultaneous pancreas-kidney transplantation independent of geography or location.

Lipid biosynthesis enzyme Agpat5 in AgRP-neurons is required for insulin-induced hypoglycemia sensing and glucagon secretion

The counterregulatory response to hypoglycemia that restores normal blood glucose levels is an essential physiological function. It is initiated, in large part, by incompletely characterized brain hypoglycemia sensing neurons that trigger the secretion of counterregulatory hormones, in particular glucagon, to stimulate hepatic glucose production. In a genetic screen of recombinant inbred BXD mice we previously identified Agpat5 as a candidate regulator of hypoglycemia-induced glucagon secretion. Here, using genetic mouse models, we demonstrate that Agpat5 expressed in agouti-related peptide neurons is required for their activation by hypoglycemia, for hypoglycemia-induced vagal nerve activity, and glucagon secretion. We find that inactivation of Agpat5 leads to increased fatty acid oxidation and ATP production and that suppressing Cpt1a-dependent fatty acid import into mitochondria restores hypoglycemia sensing. Collectively, our data show that AgRP neurons are involved in the control of glucagon secretion and that Agpat5, by partitioning fatty acyl-CoAs away from mitochondrial fatty acid oxidation and ATP generation, ensures that the fall in intracellular ATP, which triggers neuronal firing, faithfully reflects changes in glycemia.

Recurrent hypoglycemia increases hepatic gluconeogenesis without affecting glycogen metabolism or systemic lipolysis in rat

IntroductionRecurrent hypoglycemia (RH) impairs secretion of counterregulatory hormones. Whether and how RH affects responses within metabolically important peripheral organs to counterregulatory hormones are poorly understood. ObjectiveTo study the effects of RH on metabolic pathways associated with glucose counterregulation within liver, white adipose tissue and skeletal muscle. MethodsUsing a widely adopted rodent model of 3-day recurrent hypoglycemia, we first checked expression of counterregulatory hormone G-protein coupled receptors (GPCRs), their inhibitory regulators and downstream enzymes catalyzing glycogen metabolism, gluconeogenesis and lipolysis by qPCR and western blot. Then, we examined epinephrine-induced phosphorylation of PKA substrates to validate adrenergic sensitivity in each organ. Next, we measured hepatic and skeletal glycogen content, degree of breakdown by epinephrine and abundance of phosphorylated glycogen phosphorylase under hypoglycemia and that of phosphorylated glycogen synthase during recovery to evaluate glycogen turnover. Further, we performed pyruvate and lactate tolerance tests to assess gluconeogenesis. Additionally, we measured circulating FFA and glycerol to check lipolysis. The abovementioned studies were repeated in streptozotocin-induced diabetic rat model. Finally, we conducted epinephrine tolerance test to investigate systemic glycemic excursions to counterregulatory hormones. Saline-injected rats served as controls. ResultsRH increased counterregulatory hormone GPCR signaling in liver and epidydimal white adipose tissue (eWAT), but not in skeletal muscle. For glycogen metabolism, RH did not affect total content or epinephrine-stimulated breakdown in liver and skeletal muscle. Although RH decreased expression of phosphorylated glycogen synthase 2, it did not affect hepatic glycogen biosynthesis during recovery from hypoglycemia or after fasting-refeeding. For gluconeogenesis, RH upregulated fructose 1,6-bisphosphatase 1 and monocarboxylic acid transporter 1 that imports lactate as precursor, resulting in a lower blood lactate profile during hypoglycemia. In agreement, RH elevated fasting blood glucose and caused higher glycemic excursions during pyruvate tolerance test. For lipolysis, RH did not affect circulating levels of FFA and glycerol after overnight fasting or upon epinephrine stimulation. Interestingly, RH upregulated the trophic fatty acid transporter FATP1 and glucose transporter GLUT4 to increase lipogenesis in eWAT. These aforementioned changes of gluconeogenesis, lipolysis and lipogenesis were validated in streptozotocin-diabetic rats. Finally, RH increased insulin sensitivity to accelerate glucose disposal, which was attributable to upregulated visceral adipose GLUT4. ConclusionsRH caused metabolic adaptations related to counterregulation within peripheral organs. Specifically, adrenergic signaling was enhanced in liver and visceral fat, but not in skeletal muscle. Glycogen metabolism remained unchanged. Hepatic gluconeogenesis was augmented. Systemic lipolysis was unaffected, but visceral lipogenesis was enhanced. Insulin sensitivity was increased. These findings provided insights into mechanisms underlying clinical problems associated with intensive insulin therapy, such as high gluconeogenic flux and body weight gain.

Глікемічний скринінг і вторинні порушення вуглеводного обміну при ендокринній патології

Причиною поганого глікемічного контролю при цукровому діабеті може бути надлишкова секреція контрінсулярних гормонів при різних ендокринопатіях. А отже, глікемічний скринінг і скринінг ендокринних захворювань, які супроводжуються порушеннями вуглеводного обміну, повинні проводитися паралельно.

Ventromedial hypothalamic nucleus glycogen regulation of metabolic-sensory neuron AMPK and neurotransmitter expression: role of lactate.

Astrocyte glycogen is dynamically remodeled during metabolic stability and provides oxidizable l-lactate equivalents during neuroglucopenia. Current research investigated the hypothesis that ventromedial hypothalamic nucleus (VMN) glycogen metabolism controls glucostimulatory nitric oxide (NO) and/or glucoinhibitory gamma-aminobutyric acid (GABA) neuron 5'-AMP-activated protein kinase (AMPK) and transmitter marker, e.g., neuronal nitric oxide synthase (nNOS), and glutamate decarboxylase65/67 (GAD) protein expression. Adult ovariectomized estradiol-implanted female rats were injected into the VMN with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) before vehicle or l-lactate infusion. Western blot analysis of laser-catapult-microdissected nitrergic and GABAergic neurons showed that DAB caused lactate-reversible upregulation of nNOS and GAD proteins. DAB suppressed or increased total AMPK content of NO and GABA neurons, respectively, by lactate-independent mechanisms, but lactate prevented drug enhancement of pAMPK expression in nitrergic neurons. Inhibition of VMN glycogen disassembly caused divergent changes in counter-regulatory hormone, e.g. corticosterone (increased) and glucagon (decreased) secretion. Outcomes show that VMN glycogen metabolism controls local glucoregulatory transmission by means of lactate signal volume. Results implicate glycogen-derived lactate deficiency as a physiological stimulus of corticosterone release. Concurrent normalization of nitrergic neuron nNOS and pAMPK protein and corticosterone secretory response to DAB by lactate infers that the hypothalamic-pituitary-adrenal axis may be activated by VMN NO-mediated signals of cellular energy imbalance.

Neuroestradiol regulation of ventromedial hypothalamic nucleus 5'-AMP-activated protein kinase activity and counterregulatory hormone secretion in hypoglycemic male versus female rats.

Hypoglycemia activates the ultra-sensitive energy gauge 5′-AMP-activated protein kinase (AMPK) in ventromedial hypothalamic nucleus (VMN) gluco-regulatory neurons. The VMN is exemplified by high levels of expression of the enzyme aromatase, which converts testosterone to estradiol. This study examined the hypothesis that neuroestradiol imposes sex-dimorphic control of VMN AMPK activity during eu- and/or hypoglycemia. VMN tissue corresponding to distinct rostro-caudal segments was obtained by micropunch dissection from testes-intact male and estradiol-replaced ovariectomized female rats that were infused intracerebroventricularly with the aromatase inhibitor letrozole (Lz) before subcutaneous insulin (INS) injection. In euglycemic rats, Lz treatment elevated (male) or decreased (female) middle VMN phosphoAMPK content, with concurrent effects on total AMPK expression. Lz prevented hypoglycemic up-regulation of the mean pAMPK/AMPK ratio in rostral and middle segments of the male VMN, and significantly inhibited this proportion throughout the VMN of hypoglycemic female rats. Lz prevented glucagon secretion in hypoglycemic rats of each sex, and abolished hypoglycemic hypercorticosteronemia in males. Results show that neuroestradiol regulation of VMN AMPK activity during euglycemia is region-specific and gender-divergent, e.g. inhibitory in males versus stimulatory in females. Intra-VMN distribution of hypoglycemia-activated AMPK varies between sexes, but in each sex, locally-generated estradiol is critical for sensor reactivity to this stimulus. Coincident Lz attenuation of VMN AMPK and counter-regulatory hormone responses to hypoglycemia infers a possible cause-and-effect association. Further effort is needed to elucidate the cellular and molecular mechanisms that underlie sex-dimorphic neuroestradiol control of VMN total AMPK and phosphoAMPK expression during distinct metabolic states.

Adipocyte lipolysis drives acute stress-induced insulin resistance

Stress hyperglycemia and insulin resistance are evolutionarily conserved metabolic adaptations to severe injury including major trauma, burns, or hemorrhagic shock (HS). In response to injury, the neuroendocrine system increases secretion of counterregulatory hormones that promote rapid mobilization of nutrient stores, impair insulin action, and ultimately cause hyperglycemia, a condition known to impair recovery from injury in the clinical setting. We investigated the contributions of adipocyte lipolysis to the metabolic response to acute stress. Both surgical injury with HS and counterregulatory hormone (epinephrine) infusion profoundly stimulated adipocyte lipolysis and simultaneously triggered insulin resistance and hyperglycemia. When lipolysis was inhibited, the stress-induced insulin resistance and hyperglycemia were largely abolished demonstrating an essential requirement for adipocyte lipolysis in promoting stress-induced insulin resistance. Interestingly, circulating non-esterified fatty acid levels did not increase with lipolysis or correlate with insulin resistance during acute stress. Instead, we show that impaired insulin sensitivity correlated with circulating levels of the adipokine resistin in a lipolysis-dependent manner. Our findings demonstrate the central importance of adipocyte lipolysis in the metabolic response to injury. This insight suggests new approaches to prevent insulin resistance and stress hyperglycemia in trauma and surgery patients and thereby improve outcomes.

Impact of caudal hindbrain glycogen metabolism on A2 noradrenergic neuron AMPK activation and ventromedial hypothalamic nucleus norepinephrine activity and glucoregulatory neurotransmitter marker protein expression

The brain glycogen reserve is a source of oxidizable substrate fuel. Lactoprivic-sensitive hindbrain A2 noradrenergic neurons provide crucial metabolic-sensory input to downstream hypothalamic glucose-regulatory structures. Current research examined whether hindbrain glycogen fuel supply impacts A2 energy stability and governance of ventromedial hypothalamic nucleus (VMN) metabolic transmitter signaling. Male rats were injected into the caudal fourth ventricle (CV4) with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prior to continuous intra-CV4 infusion of L-lactate or vehicle. Lactate reversed DAB suppression of A2 neuron AMPK protein and up-regulated phosphoAMPK profiles. A2 dopamine-β-hydroxylase expression was refractory to DAB, but elevated by DAB/lactate. Lactate normalized A2 estrogen receptor-alpha and GPER proteins and up-regulated estrogen receptor-beta levels in DAB-treated rats. VMN norepinephrine content was decreased by DAB, but partially restored by lactate. DAB caused lactate-reversible or -irreversible augmentation of VMN glycogen phosphorylase-brain (GPbb) and -muscle type (GPmm) variant profiles, and correspondingly up- or down-regulated VMN protein markers of glucose-stimulatory nitrergic and glucose-inhibitory γ-aminobutyric acid transmission. DAB did not alter plasma glucose, but suppressed or elevated circulating glucagon and corticosterone in that order. Results show that diminished hindbrain glycogen breakdown is communicated to the VMN, in part by NE signaling, to up-regulate VMN glycogen breakdown and trigger neurochemical signaling of energy imbalance in that site. DAB effects on GPmm, VMN glycogen content, and counter-regulatory hormone secretion were unabated by lactate infusion, suggesting that aside from substrate fuel provision rate, additional indicators of glycogen metabolism such as turnover rate may be monitored in the hindbrain.

The counterregulatory response to hypoglycaemia in the pig

The domestic pig is commonly used as animal model in the pharmaceutical development of new therapeutics for treatment of diabetes. Since a formal definition of hypoglycaemia only exists in humans, the purpose of this study was to assess the counterregulatory response in the domestic pig at glucose levels known to induce symptoms of hypoglycaemia in humans. Six pigs were included in hyperinsulinaemic glucose clamps with plasma glucose targets of 2, 3 and 5mmol/L in a cross-over design, and the associated glucose counterregulatory response was assessed by measuring glucose kinetics and levels of glucagon, c-peptide, catecholamines, cortisol and growth hormone. Results showed that the 2 and 3 vs 5mmol/L clamps significantly decreased and increased the secretion of c-peptide and glucagon, respectively (P<.05). This finding was associated with increased rate of glucose appearance (Ra ) and decreased rate of glucose disappearance (Rd ) (P<.001). No marked differences in the catecholamine, growth hormone or cortisol response were observed. Consequently, like humans, pigs respond to hypoglycaemia by decreasing the pancreatic output of insulin while increasing that of glucagon, with increased glucose mobilization and decreased glucose disposal as a result. The hypoglycaemic clamps did not result in a marked secretion of the other counterregulatory hormones.

371-P: Hypoglycemia in the Brain Is Required to Augment Counterregulatory Responses to Hypoglycemia when Liver Glycogen Levels Are Enhanced

In a previous study, we showed that elevated hepatic glycogen levels can generate a signal in the liver during insulin (INS)-induced hypoglycemia that is relayed to the brain, resulting in enhanced counterregulatory (CR) hormone secretion and net hepatic glucose output (NHGO). The purpose of this study was to determine whether hypoglycemia in the brain is required for this augmentation to occur. Fasted (18 hour) dogs were studied and each experiment began with a 4h glycogen deposition period in which each animal received somatostatin and basal intraportal infusions of both INS and glucagon (GGN). Plasma glucose (PG) was doubled by an intravenous (IV) glucose infusion and liver glycogen content was enhanced in all animals (76 ± 6 and 68 ± 4 mg/g in Brain EU and HYPO, respectively) by an intraportal infusion of fructose. A 2h control period followed, during which hyperglycemia and basal replacement of hormones were continued but fructose infusion was stopped. In the final 2h hypoglycemic period, somatostatin and GGN infusions were stopped and the plasma INS level was increased 16-fold by an IV infusion of INS. During the final period, whole body PG was clamped at 52 ± 1 mg/dL in one group (HYPO; n=6). In a second group, PG in the head was clamped at 92 ± 3 mg/dL by infusing glucose into the carotid and vertebral arteries (Brain EU; n=8) while the glucose level in the rest of the body remained at 51 ± 1 mg/dL. During the final hour of the hypoglycemic period, NHGO was lower in Brain EU compared to HYPO (0.8 ± 0.2 vs. 2.2 ± 0.4 mg/kg/minute, p&amp;lt;0.01). This decrease in NHGO was associated with a decrease in CR hormone secretion, as GGN (27 ± 6 vs. 53 ± 8 pg/mL, p&amp;lt;0.01) and epinephrine (451 ± 103 vs. 1106 ± 143 pg/mL, p&amp;lt;0.01) were lower when the brain was euglycemic (Brain EU vs. HYPO, respectively). These data suggest that the augmented CR responses that occur during INS-induced hypoglycemia when liver glycogen content is enhanced, require hypoglycemia in the brain. Disclosure S.O. Warner: None. M.S. Smith: None. B. Farmer: None. J.J. Winnick: None. Funding National Institutes of Health

Hindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat

Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen- and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERβ) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERβ mediates local glycogen breakdown. DVC ERs also regulate VMN neurotransmitter signaling of energy sufficiency [γ-aminobutyric acid] or deficiency [nitric oxide, steroidogenic factor-1] during IIH. Neither hindbrain ER mediates IIH-associated diminution of VMN norepinephrine (NE) content. Both ERs oppose hypoglycemic hyperglucagonemia, while ERβ contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ERs control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -β implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.

Sex differences in forebrain estrogen receptor regulation of hypoglycemic patterns of counter-regulatory hormone secretion and ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and astrocyte glycogen metabolic enzyme expression

The female ventromedial hypothalamic nucleus (VMN) is a focal substrate for estradiol (E) regulation of energy balance, feeding, and body weight, but how E shapes VMN gluco-regulatory signaling in each sex is unclear. This study investigated the hypothesis that estrogen receptor-alpha (ERα) and/or -beta (ERβ) control VMN signals that inhibit [γ-aminobutyric acid] or stimulate [nitric oxide, steroidogenic factor-1 (SF-1)] counter-regulation in a sex-dependent manner. VMN nitrergic neurons monitor astrocyte fuel provision; here, we examined how these ER regulate astrocyte glycogen metabolic enzyme, monocarboxylate transporter, and adrenoreceptor protein responses to insulin-induced hypoglycemia (IIH) in each sex. Testes-intact male and E-replaced ovariectomized female rats were pretreated by intracerebroventricular ERα antagonist (MPP) or ERβ antagonist (PHTPP) administration before IIH. Data implicate both ER in hypoglycemic inhibition of neuronal nitric oxide synthase protein in each sex and up-regulation of glutamate decarboxylase65/67 and SF-1 expression in females. ERα and -β enhance astrocyte AMPK and glycogen synthase expression and inhibit glycogen phosphorylase in hypoglycemic females, while ERβ suppresses the same proteins in males. Differential VMN astrocyte protein responses to IIH may partially reflect ERα and -β augmentation of ERβ and down-regulation of alpha1, alpha2, and beta1 adrenoreceptor proteins in females, versus ERβ repression of GPER and alpha2 adrenoreceptor profiles in males. MPP or PHTPP pretreatment blunted counter-regulatory hormone secretion in hypoglycemic males only, suggesting that in males one or more VMN neurotransmitters exhibiting sensitivity to forebrain ER may passively regulate this endocrine outflow, whereas female forebrain ERα and -β are apparently uninvolved in these contra-regulatory responses.

Hindbrain estrogen receptor-beta antagonism normalizes reproductive and counter-regulatory hormone secretion in hypoglycemic steroid-primed ovariectomized female rats

Hindbrain dorsal vagal complex A2 noradrenergic signaling represses the pre-ovulatory luteinizing hormone (LH) surge in response to energy deficiency. Insulin-induced hypoglycemia augments A2 neuron adenosine 5′-monophosphate-activated protein kinase (AMPK) activity and estrogen receptor-beta (ERβ) expression, coincident with LH surge suppression. We hypothesized that ERβ is critical for hypoglycemia-associated patterns of LH secretion and norepinephrine (NE) activity in key reproduction-relevant forebrain structures. The neural mechanisms responsible for tight coupling of systemic energy balance and procreation remain unclear; here, we investigated whether ERβ-dependent hindbrain signals also control glucose counter-regulatory responses to hypoglycemia. Gonadal steroid-primed ovariectomized female rats were pretreated by caudal fourth ventricular administration of the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) or vehicle before insulin injection at LH surge onset. Western blot analysis of laser-microdissected A2 neurons revealed hypoglycemic intensification of AMPK activity and dopamine-β-hydroxylase protein expression; the latter response was attenuated by PHTPP pretreatment. PHTPP regularized LH release, but not preoptic GnRH-I precursor protein expression in insulin-injected rats, and reversed hypoglycemic stimulation of glucagon and corticosterone secretion. Hypoglycemia caused PHTPP-reversible changes in NE and prepro-kisspeptin protein content in the hypothalamic arcuate (ARH), but not anteroventral periventricular nucleus. Results provide novel evidence for ERβ-dependent caudal hindbrain regulation of LH and counter-regulatory hormone secretion during hypoglycemia. Observed inhibition of LH likely involves mechanisms at the axon terminal that impede GnRH neurotransmission. Data also show that caudal hindbrain ERβ exerts site-specific control of NE activity in forebrain projection sites during hypoglycemia, including the ARH where prepro-kisspeptin may be a target of that signaling.

Chromium supplementation improved post-stroke brain infarction and hyperglycemia.

Hyperglycemia is common after acute stroke and is associated with a worse outcome of stroke. Thus, a better understanding of stress hyperglycemia is helpful to the prevention and therapeutic treatment of stroke. Chromium is an essential nutrient required for optimal insulin activity and normal carbohydrate and lipid metabolism. Beyond its nutritional effects, dietary supplement of chromium causes beneficial outcomes against several diseases, in particular diabetes-associated complications. In this study, we investigated whether post-stroke hyperglycemia involved chromium dynamic mobilization in a rat model of permanent focal cerebral ischemia and whether dietary supplement of chromium improved post-stroke injury and alterations. Stroke rats developed brain infarction, hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance. Post-stroke hyperglycemia was accompanied by elevated secretion of counter-regulatory hormones including glucagon, corticosterone, and norepinephrine, decreased insulin signaling in skeletal muscles, and increased hepatic gluconeogenesis. Correlation studies revealed that counter-regulatory hormone secretion showed a positive correlation with chromium loss and blood glucose increased together with chromium loss. Daily chromium supplementation increased tissue chromium levels, attenuated brain infarction, improved hyperglycemia, and decreased plasma levels of glucagon and corticosterone in stroke rats. Our findings suggest that stroke rats show disturbance of tissue chromium homeostasis with a net loss through urinary excretion and chromium mobilization and loss might be an alternative mechanism responsible for post-stroke hyperglycemia.

Coefficient of variation of R-R interval closely correlates with glycemic variability assessed by continuous glucose monitoring in insulin-depleted patients with type 1 diabetes

AimsIn type 1 diabetic patients, insulin secretory capacity, meals and physical activity correlate with glycemic variability. Autonomic function associated with gastrointestinal motility and counterregulatory hormone secretion is another candidate which correlates with glucose variability. The aim of this study is to clarify a new clinical parameter associated with glycemic variability in insulin-depleted patients with type 1 diabetes. MethodsWe studied 31 inpatients with type 1 diabetes. We evaluated glycemic variability calculated by continuous glucose monitoring, clinical parameters and the coefficient of variation of R-R interval (CVR-R). Glycemic variability was also assessed during the daytime and nighttime. ResultsThe CVR-R showed a significant negative correlation with the whole-day standard deviation (SD) (r=−0.50, p=0.007), mean amplitude of glycemic excursions (MAGE) (r=−0.47, p=0.011), M-value (r=−0.38, p=0.048) and mean of daily differences (MODD) (r=−0.59, p=0.001). The CVR-R also showed a significant negative correlation with the nighttime SD (r=−0.59, p=0.001), MAGE (r=−0.47, p=0.011), M-value (r=−0.53, p=0.004) and MODD (r=−0.65, p=0.0003). And furthermore, the CVR-R also showed a significant negative correlation with the daytime SD (r=−0.44, p=0.019) and MAGE (r=−0.50, p=0.006), but not with the daytime M-value or MODD. The nighttime SD was significantly higher in patients with diabetic polyneuropathy than in patients without it (p=0.016), while the CVR-R was significantly lower in patients with polyneuropathy than in patients without it (p=0.009). ConclusionsCVR-R is closely correlated with glycemic variability, especially during nighttime, in insulin-depleted patients with type 1 diabetes. Measuring CVR-R may help us to presume the degree of glycemic variability in those patients.

Neural regulation of pancreatic islet cell mass and function.

Intracellular glucose signalling pathways control the secretion of glucagon and insulin by pancreatic islet α- and β-cells, respectively. However, glucose also indirectly controls the secretion of these hormones through regulation of the autonomic nervous system that richly innervates this endocrine organ. Both parasympathetic and sympathetic nervous systems also impact endocrine pancreas postnatal development and plasticity in adult animals. Defects in these autonomic regulations impair β-cell mass expansion during the weaning period and β-cell mass adaptation in adult life. Both branches of the autonomic nervous system also regulate glucagon secretion. In type 2 diabetes, impaired glucose-dependent autonomic activity causes the loss of cephalic and first phases of insulin secretion, and impaired suppression of glucagon secretion in the postabsorptive phase; in diabetic patients treated with insulin, it causes a progressive failure of hypoglycaemia to trigger the secretion of glucagon and other counterregulatory hormones. Therefore, identification of the glucose-sensing cells that control the autonomic innervation of the endocrine pancreatic and insulin and glucagon secretion is an important goal of research. This is required for a better understanding of the physiological control of glucose homeostasis and its deregulation in diabetes. This review will discuss recent advances in this field of investigation.

The Case for: Hypoglycemia Is of Cardiovascular Importance

Hypoglycemia, defined as a plasma glucose concentration <70 mg/dL, represents a major and potentially very dangerous side effect of glucose-lowering therapies in patients with diabetes, and it challenges the patient’s ability to achieve and maintain optimal glycemic control (1–3). Statistically, the incidence of mild hypoglycemia in patients with type 1 diabetes is ~30 episodes/patient/year, whereas the incidence of severe hypoglycemia (i.e., requiring third-party assistance) may be as high as 3.2 episodes/patient/year (4). Furthermore, an estimated 2–4% of deaths of people with type 1 diabetes have been attributed to hypoglycemia. On the other hand, patients with type 2 diabetes experience hypoglycemia less frequently, with an incidence of mild and severe hypoglycemic episodes of 2–10/patient/year and 0.1–0.7/patient/year, respectively (4). The occurrence of hypoglycemia, both in experimental and human settings even without diabetes or related metabolic changes, provokes substantial increase in counterregulatory hormonal secretion and affects the cardiovascular system and the brain, in turn affecting cognition, mood, and the level of consciousness. The secretion of counterregulatory hormones—of which glucagon and epinephrine are the most potent but also including norepinephrine, vasopressin, growth hormone, and cortisol—follows suppression of endogenous insulin secretion (5). In addition, a specific response that occurs within the brain includes the activation of the central sympathetic nervous system, which promotes autonomic symptoms such as sweating, tremor, a pounding heart, hunger, and anxiety (6). Severe hypoglycemia is associated with significant morbidity such as cognitive impairment and can cause major neurological disability, including difficulty concentrating, drowsiness, and poor coordination. When blood glucose falls below 2.8 mmol/L, the majority of cognitive modalities are impaired. It is essential for a patient to be aware of these neuroglycopenic symptoms because they warn him/her that prompt action is required to treat the hypoglycemia and restore blood glucose to normal. In rare cases, prolonged severe …