Screening for new inhibitors of the human Mitochondrial Pyruvate Carrier and their effects on hepatic glucose production and diabetes.

BACKGROUND: The mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production. METHODS: Here we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis. RESULTS: By screening the Prestwick compound library of 1'200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models. CONCLUSIONS: New MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production. GENERAL SIGNIFICANCE: For potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.

Mitochondrial pyruvate metabolism regulates the activation of quiescent adult neural stem cells.

Cellular metabolism is important for adult neural stem/progenitor cell (NSPC) behavior. However, its role in the transition from quiescence to proliferation is not fully understood. We here show that the mitochondrial pyruvate carrier (MPC) plays a crucial and unexpected part in this process. MPC transports pyruvate into mitochondria, linking cytosolic glycolysis to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation. Despite its metabolic key function, the role of MPC in NSPCs has not been addressed. We show that quiescent NSPCs have an active mitochondrial metabolism and express high levels of MPC. Pharmacological MPC inhibition increases aspartate and triggers NSPC activation. Furthermore, genetic Mpc1 ablation in vitro and in vivo also activates NSPCs, which differentiate into mature neurons, leading to overall increased hippocampal neurogenesis in adult and aged mice. These findings highlight the importance of metabolism for NSPC regulation and identify an important pathway through which mitochondrial pyruvate import controls NSPC quiescence and activation.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency.

Neuronal excitation imposes a high demand of ATP in neurons. Most of the ATP derives primarily from pyruvate-mediated oxidative phosphorylation, a process that relies on import of pyruvate into mitochondria occuring exclusively via the mitochondrial pyruvate carrier (MPC). To investigate whether deficient oxidative phosphorylation impacts neuron excitability, we generated a mouse strain carrying a conditional deletion of MPC1, an essential subunit of the MPC, specifically in adult glutamatergic neurons. We found that, despite decreased levels of oxidative phosphorylation and decreased mitochondrial membrane potential in these excitatory neurons, mice were normal at rest. Surprisingly, in response to mild inhibition of GABA mediated synaptic activity, they rapidly developed severe seizures and died, whereas under similar conditions the behavior of control mice remained unchanged. We report that neurons with a deficient MPC were intrinsically hyperexcitable as a consequence of impaired calcium homeostasis, which reduced M-type potassium channel activity. Provision of ketone bodies restored energy status, calcium homeostasis and M-channel activity and attenuated seizures in animals fed a ketogenic diet. Our results provide an explanation for the seizures that frequently accompany a large number of neuropathologies, including cerebral ischemia and diverse mitochondriopathies, in which neurons experience an energy deficit.

Separation and determination of cysteine enantiomers in plasma after derivatization with 4-fluoro-7-nitrobenzofurazan.

The importance of D-amino acids in mammals associated with enantio-dependent biological functions has been increasingly highlighted. In addition to naturally occurring, D-amino acid supplementation could have a positive biological impact, including cytoprotective implications. In this context, supplementation with D-cysteine has revealed beneficial effects. Quantification of cysteine enantiomers in rodent plasma has been achieved by using 4-fluoro-7-nitrobenzofurazan derivatization of the target analytes. Cystine, the main form of cysteine in the plasma, was initially reduced to cysteine using DL-dithiothreitol. Baseline enantioseparation was then achieved in less than 3 min using a (R,R)-Whelk-O 1 stationary phase and isocratic elution using CH3OH-H2O 90:10 (v/v) with 15 mM ammonium formate (apparent pH 6.0) at 0.5 mL/min. The derivatives were then detected using negative ESI-MS in SRM mode. An external calibration was employed for D-cysteine, while L-cysteine quantification, as an endogenous analyte, was addressed using a background subtraction strategy. The method was validated. Response functions were obtained from 0 to 300 µM and from 0 to 125 µM for D-cysteine and L-cysteine, respectively. The trueness ranged from 96% to 105% for both enantiomers with repeatability and intermediate precision lower than 8% and 15% for the D-form and the endogenous L-form, respectively. The method was successfully applied for determining D- and L-cysteine in mouse plasma after D-cysteine administration.

The FASTK family proteins fine-tune mitochondrial RNA processing.

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.

RNA Granules in the Mitochondria and Their Organization under Mitochondrial Stresses.

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.

Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.

Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics.

Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs)1-3. Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates4. Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

The Multifaceted Pyruvate Metabolism: Role of the Mitochondrial Pyruvate Carrier.

Pyruvate, the end product of glycolysis, plays a major role in cell metabolism. Produced in the cytosol, it is oxidized in the mitochondria where it fuels the citric acid cycle and boosts oxidative phosphorylation. Its sole entry point into mitochondria is through the recently identified mitochondrial pyruvate carrier (MPC). In this review, we report the latest findings on the physiology of the MPC and we discuss how a dysfunctional MPC can lead to diverse pathologies, including neurodegenerative diseases, metabolic disorders, and cancer.

The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments.

BACKGROUND: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. RESULTS: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. CONCLUSIONS: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

Development and validation of a chiral UHPLC-MS method for the analysis of cysteine enantiomers in biological samples.

For years, d-amino acids were thought to have a minor function in biological processes compared to that of l-enantiomers. Recently, many studies have shown that d-amino acids are present in high concentrations in microorganisms, plants, mammals and humans and execute specific biological functions. One relevant example is that of d-cysteine, whose hydrogen sulfide-producing properties have been found to protect neurons against oxidative stress and to promote dendritic development. Herein, we introduce a chiral LCMS method for the rapid determination of cysteine enantiomers under polar ionic elution conditions (MeOH/MeCN/H2O 49/49/2 v/v/v, containing 50 mM formic acid and 50 mM ammonium formate) developed on a Chiralpak® ZWIX(+) chiral stationary phase. Cysteine enantiomers were analysed in biological samples after efficient reduction of the disulfide bond in cystine; the latter was achieved with the use of 1,4-dithio-dl-threitol as a reducing agent. A baseline resolution (RS = 2.7) was obtained, and the d-enantiomer eluted before the l-enantiomer. For the enantioselective analysis, cysteine was labelled with AccQ-Tag reagent, resulting in improved chromatographic behaviour and MS detection sensitivity. The method was validated according to the Food and Drug Administration guidelines. Good linearity was determined in the ranges of 0.05-0.50 mg/L for d-cysteine and 0.11-0.56 mg/L for l-cysteine. The repeatability and intermediate precision were found to be lower than 4.0%, with trueness ranging from 95.6 to 100.2% for both enantiomers. The LOD and LOQ values were 0.02 and 0.05 mg/L for d-cysteine and 0.04 and 0.11 mg/L for l-cysteine, respectively. The method was successfully applied to cell culture samples treated with d-cysteine.

Feasibility of neurochemically profiling mouse embryonic brain and its development in utero using 1 H MRS at 14.1 T.

We aimed to evaluate the feasibility of neurochemical profiling of embryonic mouse brain developments in utero and to seek potential in vivo evidence of an energy shift in a mitochondrial pyruvate carrier 1 (MPC1) deficient mouse model. C57BL/6 embryonic mouse brains were studied in utero by anatomical MRI and short echo localized proton (1 H) MRS at 14.1 T. Two embryonic stages were studied, the energy shift (e.g., embryonic day 12.5-13, E12.5-13) and close to the birth (E17.5-18). In addition, embryonic brains devoid of MPC1 were studied at E12.5-13. The MRI provided sufficient anatomical contrasts for visualization of embryonic brain. Localized 1 H MRS offered abundant metabolites through the embryonic development from E12.5 and close to the birth, e.g., E17.5 and beyond. The abundant neurochemical information at E12.5 provided metabolic status and processes relating to cellular development at this stage, i.e., the energy shift from glycolysis to oxidative phosphorylation, evidenced by accumulation of lactate in E12.5-13 embryonic brain devoid of MPC1. The further evolution of the neurochemical profile of embryonic brains at E17.5-18 is consistent with cellular and metabolic processes towards the birth. Localized 1 H MRS study of embryonic brain development in utero is feasible, and longitudinal neurochemical profiling of embryonic brains offers valuable insight into early brain development.

In vivo stabilization of OPA1 in hepatocytes potentiates mitochondrial respiration and gluconeogenesis in a prohibitin-dependent way.

Patients with fatty liver diseases present altered mitochondrial morphology and impaired metabolic function. Mitochondrial dynamics and related cell function require the uncleaved form of the dynamin-like GTPase OPA1. Stabilization of OPA1 might then confer a protective mechanism against stress-induced tissue damages. To study the putative role of hepatic mitochondrial morphology in a sick liver, we expressed a cleavage-resistant long form of OPA1 (L-OPA1Δ) in the liver of a mouse model with mitochondrial liver dysfunction (i.e. the hepatocyte-specific prohibitin-2 knockout (Hep-Phb2-/-) mice). Liver prohibitin-2 deficiency caused excessive proteolytic cleavage of L-OPA1, mitochondrial fragmentation, and increased apoptosis. These molecular alterations were associated with lipid accumulation, abolished gluconeogenesis, and extensive liver damage. Such liver dysfunction was associated with severe hypoglycemia. In prohibitin-2 knockout mice, expression of L-OPA1Δ by in vivo adenovirus delivery restored the morphology but not the function of mitochondria in hepatocytes. In prohibitin-competent mice, elongation of liver mitochondria by expression of L-OPA1Δ resulted in excessive glucose production associated with increased mitochondrial respiration. In conclusion, mitochondrial dynamics participates in the control of hepatic glucose production.

The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state.

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.

Lethal Poisoning of Cancer Cells by Respiratory Chain Inhibition plus Dimethyl α-Ketoglutarate.

Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens.

1-Deoxydihydroceramide causes anoxic death by impairing chaperonin-mediated protein folding.

Ischaemic heart disease and stroke are the most common causes of death worldwide. Anoxia, defined as the lack of oxygen, is commonly seen in both these pathologies and triggers profound metabolic and cellular changes. Sphingolipids have been implicated in anoxia injury, but the pathomechanism is unknown. Here we show that anoxia-associated injury causes accumulation of the non-canonical sphingolipid 1-deoxydihydroceramide (DoxDHCer). Anoxia causes an imbalance between serine and alanine resulting in a switch from normal serine-derived sphinganine biosynthesis to non-canonical alanine-derived 1-deoxysphinganine. 1-Deoxysphinganine is incorporated into DoxDHCer, which impairs actin folding via the cytosolic chaperonin TRiC, leading to growth arrest in yeast, increased cell death upon anoxia-reoxygenation in worms and ischaemia-reperfusion injury in mouse hearts. Prevention of DoxDHCer accumulation in worms and in mouse hearts resulted in decreased anoxia-induced injury. These findings unravel key metabolic changes during oxygen deprivation and point to novel strategies to avoid tissue damage and death.

Mitochondria-specific photoactivation to monitor local sphingosine metabolism and function.

Photoactivation ('uncaging') is a powerful approach for releasing bioactive small-molecules in living cells. Current uncaging methods are limited by the random distribution of caged molecules within cells. We have developed a mitochondria-specific photoactivation method, which permitted us to release free sphingosine inside mitochondria and thereafter monitor local sphingosine metabolism by lipidomics. Our results indicate that sphingosine was quickly phosphorylated into sphingosine 1-phosphate (S1P) driven by sphingosine kinases. In time-course studies, the mitochondria-specific uncaged sphingosine demonstrated distinct metabolic patterns compared to globally-released sphingosine, and did not induce calcium spikes. Our data provide direct evidence that sphingolipid metabolism and signaling are highly dependent on the subcellular location and opens up new possibilities to study the effects of lipid localization on signaling and metabolic fate.

The FASTK family of proteins: emerging regulators of mitochondrial RNA biology.

The FASTK family proteins have recently emerged as key post-transcriptional regulators of mitochondrial gene expression. FASTK, the founding member and its homologs FASTKD1-5 are architecturally related RNA-binding proteins, each having a different function in the regulation of mitochondrial RNA biology, from mRNA processing and maturation to ribosome assembly and translation. In this review, we outline the structure, evolution and function of these FASTK proteins and discuss the individual role that each has in mitochondrial RNA biology. In addition, we highlight the aspects of FASTK research that still require more attention.

Efficient Mitochondrial Glutamine Targeting Prevails Over Glioblastoma Metabolic Plasticity.

Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM.Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry.Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLNHigh and GLNLow, mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLNHigh GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLNHigh cultures are CD133neg and display a mesenchymal signature in contrast to CD133pos GLNLow GBM cells.Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting. Clin Cancer Res; 23(20); 6292-304. ©2017 AACR.