Andreas Wiederkehr

Mitochondrial signals drive insulin secretion in the pancreatic β-cell

β-Cell nutrient sensing depends on mitochondrial function. Oxidation of nutrient-derived metabolites in the mitochondria leads to plasma membrane depolarization, Ca(2+) influx and insulin granule exocytosis. Subsequent mitochondrial Ca(2+) uptake further accelerates metabolism and oxidative phosphorylation. Nutrient activation also increases the mitochondrial matrix pH. This alkalinization is required to maintain elevated insulin secretion during prolonged nutrient stimulation. Together the mitochondrial Ca(2+) rise and matrix alkalinization assure optimal ATP synthesis necessary for efficient activation of the triggering pathway of insulin secretion. The sustained, amplifying pathway of insulin release also depends on mitochondrial Ca(2+) signals, which likely influence the generation of glucose-derived metabolites serving as coupling factors. Therefore, mitochondria are both recipients and generators of signals essential for metabolism-secretion coupling. Activation of these signaling pathways would be an attractive target for the improvement of β-cell function and the treatment of type 2 diabetes.

NCLX protein, but not LETM1, mediates mitochondrial Ca2+ extrusion, thereby limiting Ca2+-induced NAD(P)H production and modulating matrix redox state.

Background: Whether mitochondrial Ca2+ extrusion is mediated by NCLX (mitochondrial sodium/calcium exchanger) or LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) and controls matrix redox state is unknown. Results: NCLX, but not LETM1, increases Ca2+ extrusion, limits NAD(P)H production, and promotes matrix oxidation. Conclusion: NCLX controls the duration of matrix Ca2+ elevations and their impact on redox signaling. Significance: NCLX is a potential target for the treatment of redox-dependent diseases. Mitochondria capture and subsequently release Ca2+ ions, thereby sensing and shaping cellular Ca2+ signals. The Ca2+ uniporter MCU mediates Ca2+ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca2+ against Na+ or H+, respectively. Here we study the role of these ion exchangers in mitochondrial Ca2+ extrusion and in Ca2+-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca2+ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca2+ ([Ca2+]mt) elevations. NCLX overexpression enhanced the rates of Ca2+ efflux, whereas increasing LETM1 levels had no impact on Ca2+ extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca2+]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na+/Ca2+ exchanger inhibitor CGP37157. The [Ca2+]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na+/Ca2+ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca2+ extrusion from mitochondria. By controlling the duration of matrix Ca2+ elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca2+ signals into redox changes.

Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte–neuron lactate and ketone body shuttle systems

Medium‐chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium‐chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell‐derived human astrocytes and neurons. Using single‐cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP‐activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17‐fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.—Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium‐chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte‐neuron lactate and ketone body shuttle systems. FASEB J. 30, 1913–1926 (2016). www.fasebj.org

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

Background: Mitochondrial Ca2+ uptake affects energy metabolism and insulin secretion. Results: Knockdown of mitochondrial Ca2+ uniporter decreases respiratory chain activity and mitochondrial pH gradient generation. Conclusion: Mitochondrial Ca2+ uptake via uniporter is essential for oxidative phosphorylation and metabolism-secretion coupling. Significance: The present study identifies mechanisms of action and bioenergetic consequences of mitochondrial Ca2+ transporters in insulin-releasing cells. In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca2+ influx, which triggers insulin exocytosis. The mitochondrial Ca2+ uniporter (MCU) mediates Ca2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca2+. Suppression of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.

Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.

Mitochondrial oxidative stress mediates high-phosphate-induced secretory defects and apoptosis in insulin-secreting cells

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

Defective Mitochondrial Function and Motility Due to Mitofusin 1 Overexpression in Insulin Secreting Cells

Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal β-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.

Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells

Here, we have investigated the role of inorganic phosphate (Pi) transport in mitochondria of rat clonal β-cells. In α-toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of the phosphate transporter PiC, however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Using (31)P NMR we observed reduction of Pi pools during nutrient stimulation of INS-1E cells. Interestingly, Pi loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial Pi pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.

Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells

Mitochondria play a central role in pancreatic β‐cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non‐nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non‐nutrient secretagogues, augments mitochondrial respiration in INS‐1E (rat insulinoma cell line clone 1E) and human pancreatic β cells. The phorbol ester, phorbol 12‐myristate 13‐acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester–induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC‐dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester–stimulated INS‐1E cells maintained under resting (2.5 mM) glucose revealed a large number of phosphorylation sites that were altered during short‐term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C‐Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12‐myristate 13‐acetate–induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non‐nutrient secretagogues act, in part, via PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to β‐cell activation.—Santo‐Domingo, J., Chareyron, I., Dayon, L., Galindo, A. N., Cominetti, O., Giménez, M. P. G., De Marchi, U., Canto, C., Kussmann, M., Wiederkehr, A. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells. FASEB J. 31, 1028–1045 (2017). www.fasebj.org

A novel ATP-synthase-independent mechanism coupling mitochondrial activation to exocytosis in insulin-secreting cells

ABSTRACT Pancreatic β-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in β-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms. Highlighted Article: Secretagogues stimulating mitochondria in pancreatic β-cells act by two different mechanisms to promote insulin secretion. The pathways are distinct in their dependence on ATP-synthase, Ca2+ signals and redox changes.