Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

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 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.

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.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient ß cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among ß cells, yet testing this in vivo in the intact pancreas is challenging. Robo ßKO mice, in which the genes Robo1 and Robo2 are deleted selectively in ß cells, provide a unique model of altered islet spatial architecture without loss of ß cell differentiation or islet damage from diabetes. Combining Robo ßKO mice with intravital microscopy, we show here that Robo ßKO islets have reduced synchronized intra-islet Ca2+ oscillations among ß cells in vivo. We provide evidence that this loss is not due to a ß cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.