Leucine Suppresses α-Cell cAMP and Glucagon Secretion via a Combination of Cell-Intrinsic and Islet Paracrine Signaling.

Glucagon is critical for the maintenance of blood glucose, however nutrient regulation of pancreatic α-cells remains poorly understood. Here, we identified a role of leucine, a well-known ß-cell fuel, in the α-cell-intrinsic regulation of glucagon release. In islet perifusion assays, physiologic concentrations of leucine strongly inhibited alanine- and arginine-stimulated glucagon secretion from human and mouse islets under hypoglycemic conditions. Mechanistically, leucine dose-dependently reduced α-cell cAMP, independently of Ca2+, ATP/ADP, or fatty acid oxidation. Leucine also reduced α-cell cAMP in islets treated with somatostatin receptor 2 antagonists or diazoxide, compounds that limit paracrine signaling from ß/δ-cells. Studies in dispersed mouse islets confirmed an α-cell-intrinsic effect. The inhibitory effect of leucine on cAMP was mimicked by glucose, α-ketoisocaproate, succinate, and the glutamate dehydrogenase activator BCH and blocked by cyanide, indicating a mechanism dependent on mitochondrial metabolism. Glucose dose-dependently reduced the impact of leucine on α-cell cAMP, indicating an overlap in function; however, leucine was still effective at suppressing glucagon secretion in the presence of elevated glucose, amino acids, and the incretin GIP. Taken together, these findings show that leucine plays an intrinsic role in limiting the α-cell secretory tone across the physiologic range of glucose levels, complementing the inhibitory paracrine actions of ß/δ-cells.

Intra-islet α-cell Gs signaling promotes glucagon release.

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Leucine suppresses glucagon secretion from pancreatic islets by directly modulating α-cell cAMP.

Objective: Pancreatic islets are nutrient sensors that regulate organismal blood glucose homeostasis. Glucagon release from the pancreatic α-cell is important under fasted, fed, and hypoglycemic conditions, yet metabolic regulation of α-cells remains poorly understood. Here, we identified a previously unexplored role for physiological levels of leucine, which is classically regarded as a ß-cell fuel, in the intrinsic regulation of α-cell glucagon release. Methods: GcgCreERT:CAMPER and GcgCreERT:GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of α-cell cAMP and Ca2+ within the intact islet. Islet perifusion assays were used for simultaneous, time-resolved measurements of glucagon and insulin release from mouse and human islets. The effects of leucine were compared with glucose and the mitochondrial fuels 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH, non-metabolized leucine analog that activates glutamate dehydrogenase), α-ketoisocaproate (KIC, leucine metabolite), and methyl-succinate (complex II fuel). CYN154806 (Sstr2 antagonist), diazoxide (KATP activator, which prevents Ca2+-dependent exocytosis from α, ß, and δ-cells), and dispersed α-cells were used to inhibit islet paracrine signaling and identify α-cell intrinsic effects. Results: Mimicking the effect of glucose, leucine strongly suppressed amino acid-stimulated glucagon secretion. Mechanistically, leucine dose-dependently reduced α-cell cAMP at physiological concentrations, with an IC50 of 57, 440, and 1162 µM at 2, 6, and 10 mM glucose, without affecting α-cell Ca2+. Leucine also reduced α-cell cAMP in islets treated with Sstr2 antagonist or diazoxide, as well as dispersed α-cells, indicating an α-cell intrinsic effect. The effect of leucine was matched by KIC and the glutamate dehydrogenase activator BCH, but not methyl-succinate, indicating a dependence on mitochondrial anaplerosis. Glucose, which stimulates anaplerosis via pyruvate carboxylase, had the same suppressive effect on α-cell cAMP but with lower potency. Similarly to mouse islets, leucine suppressed glucagon secretion from human islets under hypoglycemic conditions. Conclusions: These findings highlight an important role for physiological levels of leucine in the metabolic regulation of α-cell cAMP and glucagon secretion. Leucine functions primarily through an α-cell intrinsic effect that is dependent on glutamate dehydrogenase, in addition to the well-established α-cell regulation by ß/δ-cell paracrine signaling. Our results suggest that mitochondrial anaplerosis-cataplerosis facilitates the glucagonostatic effect of both leucine and glucose, which cooperatively suppress α-cell tone by reducing cAMP.

No Increase in Kawasaki Disease-Like Illnesses During the COVID-19 Pandemic Compared With 4 Previous Years: A Medical Records Review Analysis.

BACKGROUND: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection produces a wide variety of inflammatory responses in children, including multisystem inflammatory syndrome in children, which has similar clinical manifestations as Kawasaki disease (KD). METHODS: We performed a chart review of all patients with KD-like illnesses from January 1, 2016, to May 31, 2020, at a tertiary care children's hospital within a larger health system. Relevant symptoms, comorbid illnesses, laboratory results, imaging studies, treatment, and outcomes were reviewed. Descriptive analyses to compare features over time were performed. RESULTS: We identified 81 cases of KD-like illnesses from January 1, 2016, to May 31, 2020. Few clinical features, such as gallbladder involvement, were more prevalent in 2020 than in previous years. A few patients in 2020 required more intensive treatment with interleukin 1 receptor antagonist therapy. There were no other clear differences in incidence, laboratory parameters, number of doses of intravenous immunoglobulin, or outcomes over the years of the study. CONCLUSIONS: There was no difference in incidence, laboratory parameters, or number of doses of intravenous immunoglobulin required for treatment of KD-like illnesses during the COVID-19 pandemic when compared with previous years at our institution. Kawasaki disease-like illnesses, including multisystem inflammatory syndrome in children, may not have changed substantially during the COVID-19 pandemic.

Long-term behavioral consequences of brief, repeated neonatal isolation.

Rats subjected to stressful stimuli during the stress hyporesponsive period exhibit varied neuroendocrine and behavioral changes as neonates, adolescents and adults. The current work examined the effects of neonatal isolation stress, using a within-litter design, on adult anxiety-related behavior and endocrine stress reactivity. Neonatal rats were isolated daily for 1 h from postnatal day (P) 4 to 9, a manipulation previously shown to induce hypothalamic-pituitary-adrenal (HPA) responses on P9 (Knuth, E.D., Etgen, A.M. (2005) Corticosterone secretion induced by chronic isolation in neonatal rats is sexually dimorphic and accompanied by elevated ACTH. Horm Behav 47:65-75.). Control animals were either handled briefly or left undisturbed (with-dam). Adult rats were tested for anxiety-related behavior using the elevated plus maze and open field, and for endocrine responses following restraint stress. Neonatal isolation decreased center exploration of the open field following 1 h restraint, including decreased time in the center compared to with-dam or handled controls and decreased center entries and distance traveled in the center compared to with-dam controls. It also decreased time in and entries into the open arms of the elevated plus maze compared to handled controls, suggesting enhanced anxiety-related behavior. Neonatal isolation had no effect on basal or restraint-induced levels of ACTH or corticosterone. These findings indicate that neonatal isolation may enhance anxiety-related behaviors, especially in response to stress, without altering HPA function.

Corticosterone secretion induced by chronic isolation in neonatal rats is sexually dimorphic and accompanied by elevated ACTH.

Rat pups repeatedly subjected to brief periods of isolation during the stress hyporesponsive period (SHRP) exhibit varied neuroendocrine and behavioral changes as neonates and as adults. For example, neonatal rats exhibit increased circulating corticosterone after 1-h isolation on postnatal day 9 (P9) only if they were isolated daily from P2 to P8 [McCormick, C.M., Kehoe, P., Kovacs, S., 1998. Corticosterone release in response to repeated, short episodes of neonatal isolation: evidence of sensitization. Int. J. Dev. Neurosci. 16, 175-185]. It is not known if the increase in adrenocortical response on P9 following repeated isolation is mediated by increased pituitary ACTH secretion. The present study examined the responsivity of the hypothalamic-pituitary-adrenal (HPA) axis during the SHRP following brief, repeated isolation or acute pharmacological manipulation. Removal from the nest for 1 h daily on P4-8 increased circulating corticosterone after 1-h isolation on P9 by approximately twofold. Neither unhandled nor handled controls showed a corticosterone response to 1-h isolation on P9. The increased corticosterone was sexually dimorphic, with only females showing the sensitization response. Other findings suggest that the hormonal response is centrally mediated; chronically isolated pups of both sexes exhibit increased plasma ACTH following 1-h isolation on P9. While we could not detect an increase in Fos immunoreactivity (IR) on P9 in the hypothalamic paraventricular nucleus (PVN) of chronically isolated pups, acute pharmacological activation of serotonin 2A/2C receptors produced robust activation of ACTH and corticosterone secretion as well as expression of Fos in the PVN on P9. We conclude that chronic isolation stress limited to the SHRP stimulates the neonatal HPA axis, and that the adrenal response is sexually dimorphic. In addition, PVN neurons can express Fos IR on P9 in response to a very potent activation of the HPA axis.

Neural and hormonal consequences of neonatal 5,7-dihydroxytryptamine may not be associated with serotonin depletion.

The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) is often used in neonatal rats to induce specific, rapid, and permanent depletion of brain serotonin (5-HT). One assumed benefit of using this drug in neonates is that it is well-tolerated, with pups exhibiting few side effects normally attributed to 5-HT depletion. Here, we present evidence that 5,7-DHT administered neonatally induces seizure-like behavior, decreases weight gain, and increases plasma corticosterone without depletion of brain 5-HT.