bioRxiv. 2023 Jul 31. pii: 2023.07.31.551113. [Epub ahead of print]
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 β-cell fuel, in the intrinsic regulation of α-cell glucagon release.Methods: GcgCre ERT :CAMPER and GcgCre ERT :GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of α-cell cAMP and Ca 2+ 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 (K ATP activator, which prevents Ca 2+ -dependent exocytosis from α, β, 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 IC 50 of 57, 440, and 1162 μM at 2, 6, and 10 mM glucose, without affecting α-cell Ca 2+ . 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 β/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.
Highlights: Leucine inhibits glucagon secretion from mouse and human isletsLeucine suppresses α-cell cAMP via both direct and paracrine effectsAnaplerosis via glutamate dehydrogenase is sufficient to suppress α-cell cAMPLeucine suppresses α-cell cAMP and glucagon secretion more potently than glucose.