Andrey P. Tikunov

Closure of VDAC causes oxidative stress and accelerates the Ca2+-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT.

Ribosomal protein-Mdm2-p53 pathway coordinates nutrient stress with lipid metabolism by regulating MCD and promoting fatty acid oxidation.

Significance Although progress has been made in the characterization of p53 in regulating metabolism, very little is known about the signaling pathways involved in this regulation in response to stress in vivo. Here we show that p53 controls hepatic fatty acid oxidation in mice in response to fasting. Disruption of ribosome protein (RP)-mouse double minute (Mdm)2 binding in Mdm2C305F mice results in fasting-induced hepatosteatosis. A full-dosage of p53 and an intact RP-Mdm2-p53 pathway are required for the induction of malonyl coA decarboxylase (MCD), a critical regulator of fatty acid oxidation. Thus, the RP-Mdm2-p53 pathway functions as a key regulator of hepatic lipid homeostasis in response to nutrient deprivation stress, a function that has implications in organismal survival and tumor suppression. The tumor suppressor p53 has recently been shown to regulate energy metabolism through multiple mechanisms. However, the in vivo signaling pathways related to p53-mediated metabolic regulation remain largely uncharacterized. By using mice bearing a single amino acid substitution at cysteine residue 305 of mouse double minute 2 (Mdm2C305F), which renders Mdm2 deficient in binding ribosomal proteins (RPs) RPL11 and RPL5, we show that the RP–Mdm2–p53 signaling pathway is critical for sensing nutrient deprivation and maintaining liver lipid homeostasis. Although the Mdm2C305F mutation does not significantly affect growth and development in mice, this mutation promotes fat accumulation under normal feeding conditions and hepatosteatosis under acute fasting conditions. We show that nutrient deprivation inhibits rRNA biosynthesis, increases RP–Mdm2 interaction, and induces p53-mediated transactivation of malonyl-CoA decarboxylase (MCD), which catalyzes the degradation of malonyl-CoA to acetyl-CoA, thus modulating lipid partitioning. Fasted Mdm2C305F mice demonstrate attenuated MCD induction and enhanced malonyl-CoA accumulation in addition to decreased oxidative respiration and increased fatty acid accumulation in the liver. Thus, the RP–Mdm2–p53 pathway appears to function as an endogenous sensor responsible for stimulating fatty acid oxidation in response to nutrient depletion.

Metabolomic Investigations of American Oysters Using 1H-NMR Spectroscopy

The Eastern oyster (Crassostrea virginica) is a useful, robust model marine organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic analysis to C. virginica to investigate the differences in the metabolic profile of different organ groups, and magnetic resonance imaging (MRI) to non-invasively identify the well separated organs. Metabolites were identified in perchloric acid extracts of three portions of the oyster containing: (1) adductor muscle, (2) stomach and digestive gland, and (3) mantle and gills. Osmolytes dominated the metabolome in all three organ blocks with decreasing concentration as follows: betaine > taurine > proline > glycine > ß-alanine > hypotaurine. Mitochondrial metabolism appeared most pronounced in the adductor muscle with elevated levels of carnitine facilitating ß-oxidation, and ATP, and phosphoarginine synthesis, while glycogen was elevated in the mantle/gills and stomach/digestive gland. A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions. This study identifies metabolites and corresponding 1H NMR peak assignments for future NMR-based metabolomic studies in oysters.

p53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway

Activation of p53 in response to DNA damage is essential for tumor suppression. Although previous studies have emphasized the importance of p53-dependent cell cycle arrest and apoptosis for tumor suppression, recent studies have suggested that other areas of p53 regulation, such as metabolism and DNA damage repair (DDR), are also essential for p53-dependent tumor suppression. However, the intrinsic connections between p53-mediated DDR and metabolic regulation remain incompletely understood. Here, we present data suggesting that p53 promotes nucleotide biosynthesis in response to DNA damage by repressing the expression of the phosphofructokinase-2 (PFK2) isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a rate-limiting enzyme that promotes glycolysis. PFKFB3 suppression increases the flux of glucose through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient DNA damage repair and increased cell survival. Interestingly, although p53-mediated suppression of PFKFB3 could increase the two major PPP products, NADPH and nucleotides, only nucleotide production was essential to promote DDR. By identifying the novel p53 target PFKFB3, we report an important mechanistic connection between p53-regulated metabolism and DDR, both of which play crucial roles in tumor suppression.

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans.

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the ΔgprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the ΔgprD has a much lower PKA activity upon starvation. Transcriptomics and 1H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the ΔgprB and ΔgprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in ΔgprB, while in the ΔgprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the ΔgprD strain. The 1H NMR analysis revealed significant expression of essential amino acids with elevated levels in the ΔgprD strain, compared to the wild-type and ΔgprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development.

Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth

Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae.

13C magnetic resonance spectroscopy detection of changes in serine isotopomers reflects changes in mitochondrial redox status

The glycine cleavage system (GCS), the major pathway of glycine catabolism in liver, is found only in the mitochondria matrix and is regulated by the oxidized nicotinamide adenine dinucleotide (NAD+)/reduced nicotinamide adenine dinucleotide (NADH) ratio. In conjunction with serine hydroxymethyltransferase, glycine forms the 1 and 2 positions of serine, while the 3 position is formed exclusively by GCS. Therefore, we sought to exploit this pathway to show that quantitative measurements of serine isotopomers in liver can be used to monitor the NAD+/NADH ratio using 13C NMR spectroscopy. Rat hepatocytes were treated with modulators of GCS activity followed by addition of 2‐13C‐glycine, and the changes in the proportions of newly synthesized serine isotopomers were compared to controls. Cysteamine, a competitive inhibitor of GCS, prevented formation of mitochondrial 3‐13C‐serine and 2,3‐13C‐serine isotopomers while reducing 2‐13C‐serine by 55%, demonstrating that ca. 20% of glycine‐derived serine is produced in the cytosol. Glucagon, which activates GCS activity, and the mitochondrial uncoupler carbonyl cyanide‐3‐chlorophenylhydrazone both increased serine isotopomers, whereas rotenone, an inhibitor of complex I, had the opposite effect. These results demonstrate that 13C magnetic resonance spectroscopy monitoring of the formation of serine isotopomers in isolated rat hepatocytes given 2‐13C‐glycine reflects the changes of mitochondrial redox status. Magn Reson Med, 2012.

Dose-response in a high density three-dimensional liver device with real-time bioenergetic and metabolic flux quantification

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C-labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C-glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C-glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Application of Magnetic Resonance for Metabolomic Investigation of Mollusks

Metabolites, as the end products of metabolism, represent the functional responses of a cell. Their characterization can provide insight into the underlying mechanisms of genomic or environmental actions on metabolism. The aquatic environment is varied and dynamic, providing a vast diversity of physical and chemical challenges to metabolism, making the study of the metabolites of mollusks particularly fruitful for scientists interested in comparative physiology, pharmacology and toxicology. Organ specific metabolic fingerprints can establish time dependent assessments for interpreting functional adaptations to environmental and nutritional challenges using either invasive tissue extraction from multiple individuals or non-invasive longitudinal observation of the same individual. NMR spectroscopy and magnetic resonance imaging (MRI) permit non-invasive monitoring of the metabolome.The mollusks are a useful, robust model organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton and carbon nuclear magnetic resonance (1H and 13C NMR)-based metabolomic analysis to Eastern oyster C. virginica and freshwater mussel Eastern Elliptio E. Complanata to investigate the variation in the metabolic profile of different organ groups. MRI was used to non-invasively identify the morphology of the organs. In vivo MR spectra can be obtained from single region of interest (ROI or voxel) or multiple ROI simultaneously using the technique typically called chemical shift imaging (CSI). Here we also report applications of CSI to marine samples and describe the use of the technique to study in vivo glycine metabolism in oysters using 13C MRS.A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions.

Role of voltage-dependent anion channels of the mitochondrial outer membrane in regulation of cell metabolism

The role of voltage-dependent anion channels (VDAC/porins) of the mitochondrial outer membrane in the regulation of cell metabolism is assessed using an experimental model of ethanol toxicity in cultured hepatocytes. It is demonstrated that ethanol inhibits the phosphorylating and the uncoupled mitochondrial respiration, decreases the accessibility of mitochondrial adenylate kinase in the intermembrane space, and suppresses ureagenic respiration in the cells. Treatment with digitonin at high concentrations (>80 μM)—which creates pores in the mitochondrial outer membrane, allowing bypass of closed VDAC—restores all the processes suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial function because of closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space. Our studies also reveal the role of VDAC in the regulation of liver-specific intracellular processes such as ureagenesis. The data obtained can be used in development of pharmaceuticals that would prevent VDAC closure in mitochondria of ethanol-oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.