Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic β-cells

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.

FIGURE 1.

Expression analysis and silencing of MICU1, MCU, LETM1, and UCP2 in INS-1 832/13 cells. A, detection of mRNAs of MICU1, MCU, LETM1, and UCP2 was done by reverse transcription-polymerase chain reaction using a cDNA synthesis kit followed by an amplification with conventional PCR using gene-specific primers. Amplified PCR products were electrophoretically detected on a 1% agarose gel using a 100-bp ladder. GAPDH was used as an internal control. B, efficiency of MICU1 and MCU siRNAs was validated by quantitative real time PCR. Total RNA was isolated 48 or 72 h after transfection with siRNAs against MICU1 (n = 3), MCU (n = 4), LETM1 (n = 3), and UCP2 (n = 3). GAPDH was used as a housekeeping gene. Relative mRNA expression of each gene was compared with respective controls and shown as the percentage of maximum response. ***, p < 0.001, **, p < 0.01 versus respective controls. Control Si, control siRNA.

FIGURE 2.

Silencing of MICU1, MCU, and UCP2 reduced mitochondrial Ca²⁺ uptake upon intracellular Ca²⁺ release by carbachol and ATP. INS-1 832/13 cells transiently co-transfected with mtD3cpv (mito-cameleon) and respective siRNAs were used 48 or 72 h after transfection. Cells were perfused with Ca²⁺-free EB before stimulation. A–D, left panels, mitochondrial Ca²⁺ ([Ca²⁺]_mito) was measured upon stimulation with a mixture of carbachol (Cch, 100 μm) and ATP (200 μm) in a Ca²⁺-free buffer. Curves show an average of all cells represented as a ratio of YFP/CFP over time after correction for background and photobleaching. Right panels, peak [Ca²⁺]_mito amplitude was calculated from individual curves and represented as the percentage of control. A and B, suppression of MICU1 and MCU significantly reduced [Ca²⁺]_mito (n = 9). C, LETM1 knockdown could not impact mitochondrial Ca²⁺ uptake (n = 5). D, UCP2 knockdown also reduced the [Ca²⁺]_mito (n = 13).***, p < 0.001 versus respective control. Control Si, control siRNA. ns, not significant.

FIGURE 3.

Knockdown of MICU1, MCU, and LETM1 reduced mitochondrial Ca²⁺ uptake of entering Ca²⁺ upon plasma membrane depolarization with 30 mm KCl. INS-1 832/13 cells transiently co-transfected with 4mtD3cpv (mito-cameleon) and respective siRNAs were used 48 or 72 h after transfection. A–D, left panels, average curves show a fast and transient rise in [Ca²⁺]_mito upon depolarization of cells with 30 mm KCl in the presence of extracellular Ca²⁺. Curves represent a ratio of YFP/CFP over time after correction for background and photobleaching. Right panels, peak [Ca²⁺]_mito amplitudes were calculated from individual curves and are represented as the percentage of control. A–C, silencing of MICU1 (n = 9), MCU (n = 9), and LETM1 (n = 12) significantly reduced [Ca²⁺]_mito. D, UCP2 suppression did not influence [Ca²⁺]_mito (n = 11).***, p < 0.001, **, p = 0.011 versus respective control. Control Si, control siRNA. ns, not significant.

FIGURE 4.

d-Glucose-triggered mitochondrial Ca²⁺ uptake was reduced by the knockdown of MICU1 and MCU. INS-1 832/13 cells transiently co-transfected with 4mtD3cpv (mito-cameleon) and respective siRNAs were used 48 or 72 h after transfection. Cells were first kept in 3 mm d-glucose buffer for 30 min followed by 10–15 min of incubation in d-glucose-free buffer before imaging. On the microscope, cells were perfused with d-glucose-free buffer (0G) for 3 min before switching to 16 mm d-glucose (16G) during imaging. A and B, upper and middle panels, three representative curves are shown as a ratio of YFP/CFP over time after correction for background and photobleaching and designate representative traces of [Ca²⁺]_mito from each group. Lower panels, total peak areas (A. U. = arbitrary units) were calculated from all curves and are represented as means ± S.E. Silencing of MICU1 (left lower panel; n = 12, *, p = 0.012) and MCU (right lower panel; n = 10, *, p = 0.011) significantly reduced the total peak area as compared with respective controls. Control Si, control siRNA.

FIGURE 5.

d-Glucose-induced global cytosolic Ca²⁺ signals were not affected upon silencing of MICU1 and MCU. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) was measured in INS-1 832/13 cells transiently co-transfected with D3cpv (cyto-cameleon) and respective siRNA 48 or 72 h after transfection. Cells were first kept in 3 mm d-glucose buffer followed by 10–15 min of incubation in d-glucose-free buffer (0G) before imaging. On the microscope, cells were perfused with 0G buffer for 3 min before switching to 16 mm d-glucose (16G) during acquisition. A, representative curve showing [Ca²⁺]_cyto response to 16 mm d-glucose. B, frequency of peaks per cell per min was calculated from each group at 0G and 16G. Switching of cells from 0G to 16G resulted in a significant increase in the frequency of peaks in all groups (n = 5, ***, p < 0.0001, *, p = 0.013 versus control). Frequency of peaks was not different between MICU1 and MCU siRNA-treated cells as compared with control, at both low and high d-glucose (n = 5). Control Si, control siRNA. C, the percentage of cells responding to 16 mm d-glucose was calculated from each group and was unaffected in MICU- and MCU-silenced cells (n = 5). D, Ca²⁺ peak area/3 min estimated by the addition of all values on y axis for every 3 min over the whole measurement period after normalizing the individual curves for background and photobleaching. Values in each time period were presented as -fold change as compared with basal (0–3 min). MICU1 and MCU knockdown did not affect the Ca²⁺ peak area over the whole period of measurement as compared with control (n = 5).

FIGURE 6.

d-Glucose-triggered increase in cytosolic ATP and insulin secretion was significantly reduced in MICU1 and MCU knockdown cells. Cytosolic ATP [ATP]_cyto was measured in INS-1 832/13 cells co-transfected with cytosolic luciferase and respective siRNAs. Cells were preincubated in 3 mm d-glucose (3G) before switching to 16 mm d-glucose (16G). A, peak [ATP]_cyto at 16 mm d-glucose expressed as the percentage of 3 mm glucose. Silencing of MICU1 and MCU significantly reduced cytosolic ATP (n = 32, ***, p < 0.0001,*, p = 0.032 versus control). Control Si, control siRNA. B, insulin secretion was measured in INS-1 832/13 cells 48 or 72 h after transfection with respective siRNAs. Cells were first incubated in 3 mm d-glucose for 1 h followed by a 1-h treatment with 16 mm d-glucose. GSIS was expressed as -fold change over basal upon silencing of MICU1 (n = 28; *, p < 0.05 versus control) and MCU (n = 12; ***, p < 0.0001, **, p < 0.01 versus control). C, total cellular insulin content measured from lysates of cells transfected with MICU1/MCU was not different from control (n = 3).