ABSTRACT
Glucagon is critical for the maintenance of blood glucose, however nutrient regulation of pancreatic α-cells remains poorly understood. Here, we identified a role for leucine, a well-known ß-cell fuel, in the α-cell intrinsic regulation of glucagon release. In islet perifusion assays, physiological 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 Sstr2 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 α-cell secretory tone across the physiological range of glucose levels, complementing the inhibitory paracrine actions of ß/δ-cells.
ABSTRACT
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.
