Mitochondrial Ca ²⁺ Uptake 1 (MICU1) and Mitochondrial Ca ²⁺ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic β-Cells*

Abstract

Background: The molecular contributors of the mitochondrial Ca ²⁺ uptake, which is essential for metabolism-secretion coupling in β-cells, are unknown.

Results: Knockdown of MICU1 and MCU reduced agonist- and depolarization-induced mitochondrial Ca ²⁺ sequestration, ATP production, and d-glucose-stimulated insulin secretion.

Conclusion: MICU1 and MCU are integral to metabolism-secretion coupling in β-cells.

Significance: The presented data identify MICU1 and MCU as important contributors to pancreatic β-cell function.

Abstract

In pancreatic β-cells, uptake of Ca ²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca ²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca ²⁺ uptake 1 (MICU1), mitochondrial Ca ²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca ²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca ²⁺ uptake upon both intracellular Ca ²⁺ release and Ca ²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca ²⁺ uptake in response to either intracellular Ca ²⁺ release or Ca ²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca ²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca ²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca ²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.