Nonalcoholic Fatty Liver Disease and Staging of Hepatic Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic, and it is the most common cause of liver diseases. The patients with severe insulin-resistant diabetes having high body mass index (BMI), high-grade adipose tissue insulin resistance, and high hepatocellular triacylglycerols (triglycerides; TAG) content develop hepatic fibrosis within a 5-year follow-up. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol (DAG), fatty acyl CoA, or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the "double-hit" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/nonalcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the "first hit" the hepatic concentrations of diacylglycerol increase with an increase in saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in the liver tissue of patients with NASH. Hepatocyte lipoapoptosis is a critical feature of NASH. In the "second hit," reduced glutathione levels due to oxidative stress lead to the overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused at least by two ineffectual cyclical pathways. First is the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and the second is the Kelch like-ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. In clinical practice, on ultrasonographic examination, the elevation of transaminases, γ-glutamyltransferase, and the aspartate transaminase to platelet ratio index indicates NAFLD. Fibrosis-4 index, NAFLD fibrosis score, and cytokeratin18 are used for grading steatosis, staging fibrosis, and discriminating the NASH from simple steatosis, respectively. In addition to ultrasonography, "controlled attenuation parameter," "magnetic resonance imaging proton-density fat fraction," "ultrasound-based elastography," "magnetic resonance elastography," "acoustic radiation force impulse elastography imaging," "two-dimensional shear-wave elastography with supersonic imagine," and "vibration-controlled transient elastography" are recommended as combined tests with serum markers in the clinical evaluation of NAFLD. However, to confirm the diagnosis of NAFLD, a liver biopsy is the gold standard. Insulin resistance-associated hyperinsulinemia directly accelerates fibrogenesis during NAFLD development. Although hepatocyte lipoapoptosis is a key driving force of fibrosis progression, hepatic stellate cells and extracellular matrix cells are major fibrogenic effectors. Thereby, these are pharmacological targets of therapies in developing hepatic fibrosis. Nonpharmacological management of NAFLD mainly consists of two alternatives: lifestyle modification and metabolic surgery. Many pharmacological agents that are thought to be effective in the treatment of NAFLD have been tried, but due to lack of ability to attenuate NAFLD, or adverse effects during the phase trials, the vast majority could not be licensed.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

Acetyl-CoA regulates lipid metabolism and histone acetylation modification in cancer.

Acetyl-CoA, as an important molecule, not only participates in multiple intracellular metabolic reactions, but also affects the post-translational modification of proteins, playing a key role in the metabolic activity and epigenetic inheritance of cells. Cancer cells require extensive lipid metabolism to fuel for their growth, while also require histone acetylation modifications to increase the expression of cancer-promoting genes. As a raw material for de novo lipid synthesis and histone acetylation, acetyl-CoA has a major impact on lipid metabolism and histone acetylation in cancer. More importantly, in cancer, acetyl-CoA connects lipid metabolism with histone acetylation, forming a more complex regulatory mechanism that influences cancer growth, proliferation, metastasis.

Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST).

Epigenetic modifications play diverse roles in biological systems. Nucleic acid modifications control gene expression, protein synthesis, and sensitivity to nucleic acid-cleaving enzymes. However, the mechanisms underlying the biosynthesis of nucleic acid modifications can be challenging to identify. Studying protein-ligand interactions helps decipher biosynthetic and regulatory pathways underlying biological reactions. Here, we describe a fluorescence labeling-based quantitative method for unraveling the biomolecular interactions of bacteriophage Mu DNA modification protein Mom with its ligands, using microscale thermophoresis (MST). Compared to traditional methods for studying protein-biomolecular interactions, MST requires significantly lower sample amounts, volumes, and analysis time, thus allowing screening of a large number of candidates for interaction with a protein of interest. Another distinguishing feature of the method is that it obviates the need for protein purification, often a time- and resource-consuming step, and works well with whole or partially purified cell extracts. Importantly, the method is sensitive over a broad range of molecular affinities while offering great specificity and can be used to interrogate ligands ranging from metal ions to macromolecules. Although we established this method for a DNA modification protein, it can easily be adapted to study a variety of molecular interactions engaged by proteins.

The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine.

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

mTORC2 links growth factor signaling with epigenetic regulation of iron metabolism in glioblastoma.

In cancer, aberrant growth factor receptor signaling reprograms cellular metabolism and global gene transcription to drive aggressive growth, but the underlying mechanisms are not well-understood. Here we show that in the highly lethal brain tumor glioblastoma (GBM), mTOR complex 2 (mTORC2), a critical core component of the growth factor signaling system, couples acetyl-CoA production with nuclear translocation of histone-modifying enzymes including pyruvate dehydrogenase and class IIa histone deacetylases to globally alter histone acetylation. Integrated analyses in orthotopic mouse models and in clinical GBM samples reveal that mTORC2 controls iron metabolisms via histone H3 acetylation of the iron-related gene promoter, promoting tumor cell survival. These results nominate mTORC2 as a critical epigenetic regulator of iron metabolism in cancer.

The specific molecular architecture of plant 3-hydroxy-3-methylglutaryl-CoA lyase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.

Acetyl-CoA carboxylase 1-dependent lipogenesis promotes autophagy downstream of AMPK.

Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.

ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

Modulation of matrix metabolism by ATP-citrate lyase in articular chondrocytes.

Certain dysregulated chondrocyte metabolic adaptive responses such as decreased activity of the master regulator of energy metabolism AMP-activated protein kinase (AMPK) promote osteoarthritis (OA). Metabolism intersects with epigenetic and transcriptional responses. Hence, we studied chondrocyte ATP-citrate lyase (ACLY), which generates acetyl-CoA from mitochondrial-derived citrate, and modulates acetylation of histones and transcription factors. We assessed ACLY in normal and OA human knee chondrocytes and cartilages by Western blotting and immunohistochemistry, and quantified acetyl-CoA fluorometrically. We examined histone and transcription factor lysine acetylation by Western blotting, and assessed histone H3K9 and H3K27 occupancy of iNOS, MMP3, and MMP13 promoters by chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR). We analyzed iNOS, MMP3, MMP13, aggrecan (ACAN), and Col2a1 gene expression by RT-qPCR. Glucose availability regulated ACLY expression and function, nucleocytosolic acetyl-CoA, and histone acetylation. Human knee OA chondrocytes exhibited increased ACLY activation (assessed by Ser-455 phosphorylation), associated with increased H3K9 and H3K27 acetylation. Inhibition of ACLY attenuated IL-1ß-induced transcription of iNOS, MMP3, and MMP13 by suppressing acetylation of p65 NF-κB, H3K9, and H3K27, blunted release of NO, MMP3, and MMP13, and also reduced SOX9 acetylation that promoted SOX9 nuclear translocation, leading to increased aggrecan and Col2a1 mRNA expression. ACLY is a novel player involved in regulation of cartilage matrix metabolism. Increased ACLY activity in OA chondrocytes increased nucleocytosolic acetyl-CoA, leading to increased matrix catabolism via dysregulated histone and transcription factor acetylation. Pharmacologic ACLY inhibition in OA chondrocytes globally reverses these changes and stimulates matrix gene expression and AMPK activation, supporting translational investigation in OA.

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1ß and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cytokine-inducible SH2-containing protein (cish). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation.

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.

Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

The rare sugar N-acetylated viosamine is a major component of Mimivirus fibers.

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

Specific Inhibition of Acyl-CoA Oxidase-1 by an Acetylenic Acid Improves Hepatic Lipid and Reactive Oxygen Species (ROS) Metabolism in Rats Fed a High Fat Diet.

A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, acyl-CoA oxidase-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat diet- or obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism.

Loss of Cardiolipin Leads to Perturbation of Acetyl-CoA Synthesis.

Cardiolipin (CL), the signature phospholipid of mitochondrial membranes, plays an important role in mitochondrial processes and bioenergetics. CL is synthesized de novo and undergoes remodeling in the mitochondrial membranes. Perturbation of CL remodeling leads to the rare X-linked genetic disorder Barth syndrome, which shows disparities in clinical presentation. To uncover biochemical modifiers that exacerbate CL deficiency, we carried out a synthetic genetic array screen to identify synthetic lethal interactions with the yeast CL synthase mutant crd1Δ. The results indicated that crd1Δ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Acetyl-CoA levels were decreased in the mutant. The synthesis of acetyl-CoA depends primarily on the PDH-catalyzed conversion of pyruvate in the mitochondria and on the PDH bypass in the cytosol, which synthesizes acetyl-CoA from acetate. Consistent with perturbation of the PDH bypass, crd1Δ cells grown on acetate as the sole carbon source exhibited decreased growth, decreased acetyl-CoA, and increased intracellular acetate levels resulting from decreased acetyl-CoA synthetase activity. PDH mRNA and protein levels were up-regulated in crd1Δ cells, but PDH enzyme activity was not increased, indicating that PDH up-regulation did not compensate for defects in the PDH bypass. These findings demonstrate for the first time that CL is required for acetyl-CoA synthesis, which is decreased in CL-deficient cells as a result of a defective PDH bypass pathway.

Activation of AMP-activated Protein Kinase by Metformin Induces Protein Acetylation in Prostate and Ovarian Cancer Cells.

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects.

Allosteric Regulation of Mammalian Pantothenate Kinase.

Pantothenate kinase is the master regulator of CoA biosynthesis and is feedback-inhibited by acetyl-CoA. Comparison of the human PANK3·acetyl-CoA complex to the structures of PANK3 in four catalytically relevant complexes, 5'-adenylyl-ß,γ-imidodiphosphate (AMPPNP)·Mg2+, AMPPNP·Mg2+·pantothenate, ADP·Mg2+·phosphopantothenate, and AMP phosphoramidate (AMPPN)·Mg2+, revealed a large conformational change in the dimeric enzyme. The amino-terminal nucleotide binding domain rotates to close the active site, and this allows the P-loop to engage ATP and facilitates required substrate/product interactions at the active site. Biochemical analyses showed that the transition between the inactive and active conformations, as assessed by the binding of either ATP·Mg2+ or acyl-CoA to PANK3, is highly cooperative indicating that both protomers move in concert. PANK3(G19V) cannot bind ATP, and biochemical analyses of an engineered PANK3/PANK3(G19V) heterodimer confirmed that the two active sites are functionally coupled. The communication between the two protomers is mediated by an α-helix that interacts with the ATP-binding site at its amino terminus and with the substrate/inhibitor-binding site of the opposite protomer at its carboxyl terminus. The two α-helices within the dimer together with the bound ligands create a ring that stabilizes the assembly in either the active closed conformation or the inactive open conformation. Thus, both active sites of the dimeric mammalian pantothenate kinases coordinately switch between the on and off states in response to intracellular concentrations of ATP and its key negative regulators, acetyl(acyl)-CoA.

Retinal Hypercholesterolemia Triggers Cholesterol Accumulation and Esterification in Photoreceptor Cells.

The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1(-/-)Cyp46a1(-/-) mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1(-/-)Cyp46a1(-/-) retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in human OS. We suggest a mechanism for cholesterol ester accumulation in the OS and that activity impairment of ACAT1 in humans may underlie the development of subretinal drusenoid deposits, a hallmark of age-related macular degeneration, which is a common blinding disease. We generated Cyp27a1(-/-)Cyp46a1(-/-)Acat1(-/-) mice, characterized their retina by different imaging modalities, and confirmed that unesterified cholesterol does accumulate in their OS and that there is photoreceptor apoptosis and OS degeneration in this line. Our results provide insights into the retinal response to local hypercholesterolemia and the retinal significance of cholesterol esterification, which could be cell-specific and both beneficial and detrimental for retinal structure and function.