Mitochondrial Pyruvate Carrier Research Articles

Therapeutic potential of deuterium-stabilized (R)-pioglitazone-PXL065-for X-linked adrenoleukodystrophy.

X‐linked adrenoleukodystrophy (ALD) results from ABCD1 gene mutations which impair Very Long Chain Fatty Acids (VLCFA; C26:0 and C24:0) peroxisomal import and β‐oxidation, leading to accumulation in plasma and tissues. Excess VLCFA drives impaired cellular functions (e.g. disrupted mitochondrial function), inflammation, and neurodegeneration. Major disease phenotypes include: adrenomyeloneuropathy (AMN), progressive spinal cord axonal degeneration, and cerebral ALD (C‐ALD), inflammatory white matter demyelination and degeneration. No pharmacological treatment is available to‐date for ALD. Pioglitazone, an anti‐diabetic thiazolidinedione, exerts potential benefits in ALD models. Its mechanisms are genomic (PPARγ agonism) and nongenomic (mitochondrial pyruvate carrier—MPC, long‐chain acyl‐CoA synthetase 4—ACSL4, inhibition). However, its use is limited by PPARγ‐driven side effects (e.g. weight gain, edema). PXL065 is a clinical‐stage deuterium‐stabilized (R)‐enantiomer of pioglitazone which lacks PPARγ agonism but retains MPC activity. Here, we show that incubation of ALD patient‐derived cells (both AMN and C‐ALD) and glial cells from Abcd1‐null mice with PXL065 resulted in: normalization of elevated VLCFA, improved mitochondrial function, and attenuated indices of inflammation. Compensatory peroxisomal transporter gene expression was also induced. Additionally, chronic treatment of Abcd1‐null mice lowered VLCFA in plasma, brain and spinal cord and improved both neural histology (sciatic nerve) and neurobehavioral test performance. Several in vivo effects of PXL065 exceeded those achieved with pioglitazone. PXL065 was confirmed to lack PPARγ agonism but retained ACSL4 activity of pioglitazone. PXL065 has novel actions and mechanisms and exhibits a range of potential benefits in ALD models; further testing of this molecule in ALD patients is warranted.

Downregulation of mitochondrial pyruvate carrier 2 aggravates neuronal injury in the cortex following cerebral ischemia in rat

Cerebral ischemia is one of the most common disabling and lethal diseases worldwide, but its underlying mechanisms remain unclear. Mitochondrial pyruvate carrier 2 (MPC2), a subunit of MPC complex, plays pivotal roles in coordinating glycolytic and mitochondrial activities. In the present study, the expression of MPC2 was significantly reduced in the ischemic cerebral cortex of rats at 24 h after bilateral internal carotid artery occlusion (BICAO), and in the cortical neurons after 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation treatment. After MPC2 gene knockdown, the number and expression of neurons were remarkably decreased in the ischemic cerebral cortex of BICAO rats and OGD-treated neurons. UK5099 significantly reduced the number, expression and viability of OGD-treated neurons, and resulted in a significant decrease in length of neurite. Using RNA-sequencing (RNA-seq) technique, we further identified MPC2-related differential genes in the ischemic cerebral cortex of BICAO rats. In conclusion, our results suggested that the decrease in MPC2 expression aggravated ischemic injury, and MPC2-related genes might be a novel therapeutic target for cerebral ischemia.

Lactate Maintains BCR/Abl Expression and Signaling in Chronic Myeloid Leukemia Cells Under Nutrient Restriction.

This study was directed to deepen the effects of nutrient shortage on BCR/Ablprotein expression and signaling in chronic myeloid leukemia (CML) cells. The backbone of the study was cell culture in medium lacking glucose, the consumption of which we had previously shown to drive BCR/Ablprotein suppression, and glutamine, the other main nutrient besides glucose. In this context, we focused on the role of lactate, the main by-product of glucose metabolism under conditions of rapid cell growth, in particular as a modulator of the maintenance of CML stem/progenitor cell potential, a crucial determinant of disease course and relapse of disease. The results obtained indicated that lactate is a powerful surrogate of glucose to prevent the suppression of BCR/Abl signaling and is therefore capable to maintain BCR/Abl-dependent CML stem/progenitor cell potential. A number of metabolism-related functional and phenotypical features of CML cells were also determined. Among these, we focused on the effect of lactate on oxygen consumption rate, the dependence of this effect on the cell surface lactate carrier MCT-1, and the relationship of the lactate effect to pyruvate and to the activity of mitochondrial pyruvate carrier.

Targeting temporal metabolic needs of T follicular helper cells to treat cGVHD utilizing a mitochondrial pyruvate carrier inhibitor

Abstract The curative potential of allogeneic hematopoietic stem cell transplantation is limited by development of chronic graft versus host disease (cGVHD) and metabolic regulation of immune cells is a promising therapeutic target. During cGVHD, interactions between T follicular helper (TFH) and germinal center B (GCB) cells result in production of alloreactive class-switched antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. We show the metabolic dependencies of TFH change throughout cGVHD progression and targeting mitochondrial pyruvate transport is an effective strategy to mitigate disease. Pyruvate, the end product of glycolysis, is transported by the mitochondrial pyruvate carrier (MPC) for use in the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). TFH require glycolysis in order to induce a GC reaction, both in an immunization model and in an in vivo systemic cGVHD model with bronchiolitis obliterans (BO). Furthermore, Seahorse data from TFH extracted early in disease development in the BO model demonstrate increased dependency on glycolysis. But Seahorse data also indicates TFH undergo a shift away from glycolysis and toward OXPHOS as cGVHD continues. Based on the central role of MPC in tying pyruvate to multiple metabolic pathways, we examined an inhibitor of MPC (7ACC1) in reducing TFH activity. Administration of 7ACC1 resulted in inhibition of GCB production of class switched antibody in an in vitro assay. These findings were additionally supported in vivo, whereby daily treatment after establishment of cGVHD resulted in a decrease in disease severity. These data provide evidence that inhibiting mitochondrial pyruvate transport is a viable therapeutic strategy for cGVHD. This work was supported by grants from National Institutes of Health, National Institute of Allergy, and Infectious Diseases R37 AI34495, T32 AI007313; National Heart, Lung, and Blood Institute R01 HL56067, R01 HL11879, R01 HL115114 ; National Cancer Institute P01 CA065493

Mitochondrial oxidative phosphorylation fuels endothelial control of blood vessel tone

Arteries and veins are lined by non‐proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy‐demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully‐formed vascular wall.Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation at mitochondrial complex V disrupts calcium‐dependent, nitric oxide‐mediated endothelial cell control of vascular tone. Basal, mechanically‐activated, and agonist‐evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for endothelial cell energy production is independent of species, sex, or vascular bed.These data show that mitochondrial ATP is necessary for the obligatory role of endothelial cells in the control of blood vessel diameter, and validate the idea of targeting endothelial cell metabolism to treat endothelial cell dysfunction in cardiovascular disease.

A New Method to Analyse Myocardial Substrate Metabolism and Mitochondrial Function in Intact Cardiac Tissue Slices

The progression of cardiac diseases is often accompanied by disturbances in substrate metabolism. Extracellular flux analysis has become a standard tool to investigate metabolic alterations in cell lines. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes as well as the cultivation procedure that is required for cell attachment to the cell culture plates might affect metabolism. Therefore, we developed a flux analyser‐based method to measure substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof‐of‐principle approach in remote myocardium after myocardial infarction.To yield cardiac tissue pieces of comparable size, mouse cardiac tissue was sliced (150 µm) using a vibratome, and tissue pieces (diameter 1.9 mm) of these slices were punched out. Using “islet capture plates” in a Seahorse XFe 24 analyser, oxygen consumption rates (OCR) were measured at baseline and after FCCP‐induced uncoupling in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long‐chain fatty acid metabolism, CPT1 was inhibited by etomoxir, and Glc/Gln metabolism by inhibition of mitochondrial pyruvate carrier (MPC) and glutaminase (Gls) with UK5099/BPTES. Optical mapping of membrane potential was used to assess action potentials in tissue slices as indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia and reperfusion (n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two‐sample t‐test.Basal OCR was 53±8 pmol/min, and FCCP increased OCR to 92±18 pmol/min. Both etomoxir and UK5099/BPTES reduced OCR indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed regular action potential characteristics and propagation. After myocardial infarction, CPT1 inhibition caused a smaller reduction of uncoupled mitochondrial OCR in I/R animals compared to sham (40±13 vs. 52±4%, p<0.05). This effect was caused by an increased metabolism of Glc/Glu (37±13 vs. 24±4 pmol/min, p<0.05), whilst the effect of CPT1 was not different.Here, we describe a new method to analyse cardiac metabolism using cardiac tissue slices that metabolise fatty acids as well as glucose, and show high functional integrity. Therefore, this method has the potential to expand the methodological alternatives to investigate cardiac substrate metabolism. In a proof‐of‐principle approach, the analysis of cardiac substrate metabolism of the remote myocardium after I/R showed an augmented glucose/glutamine metabolism.

Simultaneously targeting hyperinflammation and hyperglycemia following respiratory viral infection

Abstract Individuals with hyperglycemia are at great risk of developing exaggerated pulmonary inflammation following infection with respiratory viruses including SARS-CoV-2. Conversely, SARS-CoV-2 infection has been linked to the development of new onset of hyperglycemia and diabetes. Despite global vaccination efforts, SARS-CoV-2 infection continues to be a great public health challenge, and corticosteroids targeting hyper-inflammation remain the mainstay treatment for severe COVID-19. However, steroidal medicine arises the risk of hyperglycemia, limiting its usage. Here, we showed that a second-generation thiazolidines drug MSDC-0602K (MSDC), a mitochondrial pyruvate carrier inhibitor, simultaneously decreased excessive pulmonary inflammation and hyperglycemia in normal and obese hosts during influenza infection, as such, reducing host morbidity and mortality. scRNA-seq analysis revealed that MSDC treatment reduced lung macrophage inflammation, and myeloid-specific ablation of MPC2 led to dampened lung inflammation. Further, MSDC inhibited human lung macrophage inflammatory responses following SARS-CoV-2 infection, and dampened inflammation in lung autopsies of COVID-19 patients. Mechanistically, MSDC treatment destabilized HIF-1a protein, a transcription factor promoting inflammatory responses of lung macrophages, by repressing cellular Acetyl-CoA levels and HIF-1a acetylation. Our results suggest that MSDC has the great potential to treat severe COVID-19 or other respiratory viral infection, particularly in patients with underlying metabolic conditions, by simultaneously repressing hyper-cytokinemia and hyperglycemia. Supported by NIH AG069264, NIH AI150599

The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function

SummaryGlycolysis, including both lactate fermentation and pyruvate oxidation, orchestrates CD8+ T cell differentiation. However, how mitochondrial pyruvate metabolism and uptake controlled by the mitochondrial pyruvate carrier (MPC) impact T cell function and fate remains elusive. We found that genetic deletion of MPC drives CD8+ T cell differentiation toward a memory phenotype. Metabolic flexibility induced by MPC inhibition facilitated acetyl-coenzyme-A production by glutamine and fatty acid oxidation that results in enhanced histone acetylation and chromatin accessibility on pro-memory genes. However, in the tumor microenvironment, MPC is essential for sustaining lactate oxidation to support CD8+ T cell antitumor function. We further revealed that chimeric antigen receptor (CAR) T cell manufacturing with an MPC inhibitor imprinted a memory phenotype and demonstrated that infusing MPC inhibitor-conditioned CAR T cells resulted in superior and long-lasting antitumor activity. Altogether, we uncover that mitochondrial pyruvate uptake instructs metabolic flexibility for guiding T cell differentiation and antitumor responses.

Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones. MethodsWe recombinantly expressed and purified human MPC hetero-complexes and studied their composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling. ResultsWe determined that the functional unit of human MPC is a hetero-dimer. We compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrated that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identified 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects. ConclusionsThis work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.

Mitochondrial pyruvate carrier blockade results in decreased osteoclastogenesis and bone resorption via regulating mitochondrial energy production

It’s widely accepted that increasing mitochondrial respiration plays a pivotal role during osteoclastogenesis. Mitochondrial pyruvate carrier (MPC) is the key transporter that links glycolysis to mitochondrial respiration but little is known about its role during osteoclastogenesis. Our goal was to determine the effects of its blockade on osteoclastogenesis and bone resorption in vivo and in vitro. To address this issue, we performed gene knockdown or pharmacologically inhibited MPC in primary bone marrow-derived monocytes (BMMs) or in an ovariectomized mouse model. We also studied the metabolic changes in RANKL-induced differentiating BMMs with MPC blockade and performed rescue experiments. We found that MPC blockade resulted in decreased osteoclastogenesis both in vivo and in vitro and inhibiting MPC significantly alleviated ovariectomy-induced trabecular bone loss. Further investigations showed that MPC blockade significantly reversed the metabolic profile related to RANK activation, including decreased intermediates involved in citric acid cycle and glutamine metabolism. Moreover, metabolic flux analysis verified that MPC blockade decreased pyruvate flux into TCA cycle with no significant effect on glycolysis. Besides, MPC blockade resulted in suppressed mitochondrial biogenesis in addition to oxidative phosphorylation. Rescue experiments revealed that inhibiting pyruvate dehydrogenase kinase (PDK) via sodium dichloroacetate (DCA), but not targeting glutamine metabolism, could reverse the effects of MPC blockade on osteoclastogenesis. These implied that the effects of MPC blockade were mediated by reduced pyruvate fuel into citric acid cycle in multiple aspects. Taken together, our data demonstrated the inhibitory effects of MPC blockade on osteoclastogenesis, which was mediated by decreased mitochondrial energy production.

Reprogramming hormone-sensitive prostate cancer to a lethal neuroendocrine cancer lineage by mitochondrial pyruvate carrier (MPC)

Cell lineage reprogramming is the main approach for cancer cells to acquire drug resistance and escape targeted therapy. The use of potent targeted therapies in cancers has led to the development of highly aggressive carcinoma, including neuroendocrine prostate cancer (NEPC). Although metabolic reprogramming has been reported to be essential for tumor growth and energy production, the relationship between metabolic reprogramming and lineage differentiation which can cause hormone therapy resistance has never been reported in prostate cancer (PCa). Moreover, as there is still no efficient therapy for NEPC, it is urgent to reverse this lineage differentiation during the hormone therapy. Here for the first time, we used in vitro and in vivo human PCa models to study the effect of metabolic reprogramming on the lineage differentiation from the androgen receptor (AR)–dependent adenocarcinoma to AR-independent NEPC. This lineage differentiation leads to antiandrogen drug resistance and tumor development. This phenotype is enabled by the loss of mitochondrial pyruvate carrier (MPC), the gate for mitochondrial pyruvate influx, and can be reversed by MPC overexpression. Morphologic and cellular studies also demonstrate that the pyruvate kinase M2 (PKM2) involved epithelium–mesenchymal transition process mediated this lineage alteration. Its inhibition is a potential treatment for MPC-lo tumors. All of these results suggest that metabolic rewiring can act as a starter for increased cellular plasticity which leads to antiandrogen therapy resistance through lineage differentiation. This study provides us with a potent treatment target for therapy-induced, enzalutamide-resistant NE-like prostate cancer.

A Mechanical Bridge to Recovery as a Bridge to Discovery: Learning From Few and Applying to Many.

A Mechanical Bridge to Recovery as a Bridge to Discovery: Learning From Few and Applying to Many.

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.

Identification of Four Metabolic Subtypes of Glioma Based on Glycolysis-Cholesterol Synthesis Genes.

Based on alterations in gene expression associated with the production of glycolysis and cholesterol, this research classified glioma into prognostic metabolic subgroups. In this study, data from the CGGA325 and The Cancer Genome Atlas (TCGA) datasets were utilized to extract single nucleotide variants (SNVs), RNA-seq expression data, copy number variation data, short insertions and deletions (InDel) mutation data, and clinical follow-up information from glioma patients. Glioma metabolic subtypes were classified using the ConsensusClusterPlus algorithm. This study determined four metabolic subgroups (glycolytic, cholesterogenic, quiescent, and mixed). Cholesterogenic patients had a higher survival chance. Genome-wide investigation revealed that inappropriate amplification of MYC and TERT was associated with improper cholesterol anabolic metabolism. In glioma metabolic subtypes, the mRNA levels of mitochondrial pyruvate carriers 1 and 2 (MPC1/2) presented deletion and amplification, respectively. Differentially upregulated genes in the glycolysis group were related to pathways, including IL-17, HIF-1, and TNF signaling pathways and carbon metabolism. Downregulated genes in the glycolysis group were enriched in terpenoid backbone biosynthesis, nitrogen metabolism, butanoate metabolism, and fatty acid metabolism pathway. Cox analysis of univariate and multivariate survival showed that risks of glycolysis subtypes were significantly higher than other subtypes. Those results were validated in the CGGA325 dataset. The current findings greatly contribute to a comprehensive understanding of glioma and personalized treatment.

Identification of Novel Mitochondrial Pyruvate Carrier Inhibitors by Homology Modeling and Pharmacophore-Based Virtual Screening.

The mitochondrial pyruvate carrier (MPC) is an inner-mitochondrial membrane protein complex that has emerged as a drug target for treating a variety of human conditions. A heterodimer of two proteins, MPC1 and MPC2, comprises the functional MPC complex in higher organisms; however, the structure of this complex, including the critical residues that mediate binding of pyruvate and inhibitors, remain to be determined. Using homology modeling, we identified a putative substrate-binding cavity in the MPC dimer. Three amino acid residues (Phe66 (MPC1) and Asn100 and Lys49 (MPC2)) were validated by mutagenesis experiments to be important for substrate and inhibitor binding. Using this information, we developed a pharmacophore model and then performed a virtual screen of a chemical library. We identified five new non-indole MPC inhibitors, four with IC50 values in the nanomolar range that were up to 7-fold more potent than the canonical inhibitor UK-5099. These novel compounds possess drug-like properties and complied with Lipinski’s Rule of Five. They are predicted to have good aqueous solubility, oral bioavailability, and metabolic stability. Collectively, these studies provide important information about the structure-function relationships of the MPC complex and for future drug discovery efforts targeting the MPC.

Identification and characterization of novel MPC1 gene variants causing mitochondrial pyruvate carrier deficiency.

Pyruvate, the end product of glycolysis, is a key metabolic molecule enabling mitochondrial adenosine triphosphate synthesis and takes part in multiple biosynthetic pathways within mitochondria. The mitochondrial pyruvate carrier (MPC) plays a vital role in transporting pyruvate from the cytosol into the organelle. In humans, MPC is a hetero-oligomeric complex formed by the MPC1 and MPC2 paralogs that are both necessary to stabilize each other and form a functional MPC. MPC deficiency (OMIM#614741) due to pathogenic MPC1 variants is a rare autosomal recessive disease involving developmental delay, microcephaly, growth failure, and increased serum lactate and pyruvate. To date, two MPC1 variants in four cases have been reported, though only one with a detailed clinical description. Herein, we report three novel pathogenic MPC1 variants in six patients from three unrelated families, identified within European, Kuwaiti, and Chinese mitochondrial disease patient cohorts, one of whom presented as a Leigh-like syndrome. Functional analysis in primary fibroblasts from the patients revealed decreased expression of MPC1 and MPC2. We rescued pyruvate-driven oxygen consumption rate in patient's fibroblasts by reconstituting with wild-type MPC1. Complementing homozygous MPC1 mutant cDNA with CRISPR-deleted MPC1 C2C12 cells verified the mechanism of variants: unstable MPC complex or ablated pyruvate uptake activity. Furthermore, we showed that glutamine and beta-hydroxybutyrate were alternative substrates to maintain mitochondrial respiration when cells lack pyruvate. In conclusion, we expand the clinical phenotypes and genotypes associated with MPC deficiency, with our studies revealing glutamine as a potential therapy for MPC deficiency.

Peroxisomal support of mitochondrial respiratory efficiency promotes ER stress survival.

Endoplasmic reticulum stress (ERS) occurs when cellular demand for protein folding exceeds the capacity of the organelle. Adaptation and cell survival in response to ERS requires a critical contribution by mitochondria and peroxisomes. During ERS responses, mitochondrial respiration increases to ameliorate reactive oxygen species (ROS) accumulation. We now show in yeast that peroxisome abundance also increases to promote an adaptive response. In pox1Δ cells, which are defective in peroxisomal β-oxidation of fatty acids, the respiratory response to ERS is impaired and ROS accrues. However, the respiratory response to ERS is rescued and ROS production is mitigated in pox1Δ cells overexpressing Mpc1, the mitochondrial pyruvate carrier that provides another source of acetyl CoA to fuel the tricarboxylic acid cycle and oxidative phosphorylation. Using proteomics, select mitochondrial proteins were identified that undergo upregulation upon ERS to remodel the respiratory machinery. The abundance of several peroxisome-based proteins was also increased, corroborating the role of peroxisomes in ERS adaptation. Finally, ERS stimulates assembly of respiratory complexes into higher-order supercomplexes, underlying increased electron transfer efficiency. Our results highlight peroxisomal and mitochondrial support for ERS adaptation to favor cell survival.

Mitochondrial pyruvate carrier inhibitors improve metabolic parameters in diet-induced obese mice

The mitochondrial pyruvate carrier (MPC) is an inner mitochondrial membrane complex that plays a critical role in intermediary metabolism. Inhibition of the MPC, especially in liver, may have efficacy for treating type 2 diabetes mellitus. Herein, we examined the antidiabetic effects of zaprinast and 7ACC2, small molecules which have been reported to act as MPC inhibitors. Both compounds activated a bioluminescence resonance energy transfer–based MPC reporter assay (reporter sensitive to pyruvate) and potently inhibited pyruvate-mediated respiration in isolated mitochondria. Furthermore, zaprinast and 7ACC2 acutely improved glucose tolerance in diet-induced obese mice in vivo. Although some findings were suggestive of improved insulin sensitivity, hyperinsulinemic–euglycemic clamp studies did not detect enhanced insulin action in response to 7ACC2 treatment. Rather, our data suggest acute glucose-lowering effects of MPC inhibition may be due to suppressed hepatic gluconeogenesis. Finally, we used reporter sensitive to pyruvate to screen a chemical library of drugs and identified 35 potentially novel MPC modulators. Using available evidence, we generated a pharmacophore model to prioritize which hits to pursue. Our analysis revealed carsalam and six quinolone antibiotics, as well as 7ACC1, share a common pharmacophore with 7ACC2. We validated that these compounds are novel inhibitors of the MPC and suppress hepatocyte glucose production and demonstrated that one quinolone (nalidixic acid) improved glucose tolerance in obese mice. In conclusion, these data demonstrate the feasibility of therapeutic targeting of the MPC for treating diabetes and provide scaffolds that can be used to develop potent and novel classes of MPC inhibitors.

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

Abstract 9124: The Role of Mitochondrial Pyruvate Carrier on Myocardial Salvage Following Acute Myocardial Infarction

Acute myocardial infarction (AMI) may progress to heart failure (HF). Ischemic-reperfusion injury (I/R) accounts for ~50% of the damage post-AMI. Currently, there are minimal interventions to mitigate I/R and prevent HF. Mitochondrial Pyruvate Carrier (MPC) is considered cardio-protective, and deletion causes HF. The purpose of this investigation was to determine if cardiac MPC deletion (MPC -/- ) in mice limits myocardial salvage following I/R. We hypothesized MPC -/- mice would have more necrosis and less myocardial salvage compared to wild type (WT) littermates.MPC1 was targeted in C57Bl/6 mice by placing loxP sites in the introns flanking the exon 3-5 of MPC1. A cardiac-specific deletion was used by intraperitoneal injections of tamoxifen at 8 weeks old. Four weeks post injection, prior to development of HF, mice were intubated, ventilated, and the left anterior descending artery was occluded for 30 minutes then reperfused for 2 hours. Next, Evans-Blue was injected into the heart to define the ischemic area. Hearts of paired littermates between WT (n=6) and MPC -/- (n=6) were excised and cut into 1-mm transverse slices. Slices were stained with triphenyltetrazolium-chloride (TTC) to define myocardial salvage. Next, slices were imaged and quantified by independent researchers using planimetry. Paired t-tests with an α-level of 0.05 were used to compare groups. Litters not subjected to I/R showed significant (p<0.001) MPC1 deletion (WT: 0.28±0.11 au vs. MPC -/- : 0.06±0.02 au). The area at risk (AAR) was comparable (p=0.12) between groups (WT: 46.7±5.9% vs. MPC -/- : 54.3±13.3), indicating equivalent coronary ligation-induced ischemia. Within the AAR, MPC -/- mice showed significantly (p=0.04) more necrosis (MPC -/- : 52.8±10.4% vs. WT: 42.7±17.4%) with less myocardial salvage (MPC -/- : 47.2±10.4% vs. WT: 57.3±10.6%). MPC -/- mice also had significantly (p=0.002) more absolute necrosis (28.4±7.8%) compared to WT mice (19.8±7.8%). There were no significant differences (p>0.05) in percent viable or salvageable tissue in relation to total area. In conclusion, following I/R, MPC -/- mice showed more absolute and standardized necrosis with less myocardial salvage, suggesting when cardiac MPC is reduced it causes more myocardial damage leading to HF.