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

Objective: Pancreatic islets are nutrient sensors that regulate organismal blood glucose homeostasis. Glucagon release from the pancreatic alpha-cell is important under fasted, fed, and hypoglycemic conditions, yet metabolic regulation of alpha-cells remains poorly understood. Here, we identified a previously unexplored role for physiological levels of leucine, which is classically regarded as a beta-cell fuel, in the intrinsic regulation of alpha-cell glucagon release. Methods: GcgCreERT:CAMPER and GcgCreERT:GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of alpha-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), alpha-ketoisocaproate (KIC, leucine metabolite), and methyl-succinate (complex II fuel). CYN154806 (Sstr2 antagonist), diazoxide (KATP activator, which prevents Ca2+-dependent exocytosis from alpha, beta, and delta-cells), and dispersed alpha-cells were used to inhibit islet paracrine signaling and identify alpha-cell intrinsic effects. Results: Mimicking the effect of glucose, leucine strongly suppressed amino acid-stimulated glucagon secretion. Mechanistically, leucine dose-dependently reduced alpha-cell cAMP at physiological concentrations, with an IC50 of 57, 440, and 1162 uM at 2, 6, and 10 mM glucose, without affecting alpha-cell Ca2+. Leucine also reduced alpha-cell cAMP in islets treated with Sstr2 antagonist or diazoxide, as well as dispersed alpha-cells, indicating an alpha-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 alpha-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 alpha-cell cAMP and glucagon secretion. Leucine functions primarily through an alpha-cell intrinsic effect that is dependent on glutamate dehydrogenase, in addition to the well-established alpha-cell regulation by beta/delta-cell paracrine signaling. Our results suggest that mitochondrial anaplerosis-cataplerosis facilitates the glucagonostatic effect of both leucine and glucose, which cooperatively suppress alpha-cell tone by reducing cAMP.