Cellular metabolic and autophagic pathways: Traffic control by redox signaling

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy–lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function.     

Pyruvate fuels mitochondrial respiration and proliferation of breast cancer cells: effect of monocarboxylate transporter inhibition

Recent studies have highlighted the fact that cancer cells have an altered metabolic phenotype, and this metabolic reprogramming is required to drive the biosynthesis pathways necessary for rapid replication and proliferation. Specifically, the importance of citric acid cycle-generated intermediates in the regulation of cancer cell proliferation has been recently appreciated. One function of MCTs (monocarboxylate transporters) is to transport the citric acid cycle substrate pyruvate across the plasma membrane and into mitochondria, and inhibition of MCTs has been proposed as a therapeutic strategy to target metabolic pathways in cancer. In the present paper, we examined the effect of different metabolic substrates (glucose and pyruvate) on mitochondrial function and proliferation in breast cancer cells. We demonstrated that cancer cells proliferate more rapidly in the presence of exogenous pyruvate when compared with lactate. Pyruvate supplementation fuelled mitochondrial oxygen consumption and the reserve respiratory capacity, and this increase in mitochondrial function correlated with proliferative potential. In addition, inhibition of cellular pyruvate uptake using the MCT inhibitor α-cyano-4-hydroxycinnamic acid impaired mitochondrial respiration and decreased cell growth. These data demonstrate the importance of mitochondrial metabolism in proliferative responses and highlight a novel mechanism of action for MCT inhibitors through suppression of pyruvate-fuelled mitochondrial respiration.     

CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis

Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole‐body CLPP‐deficient mice are protected from diet‐induced obesity and insulin resistance, which was not present in mouse models with either liver‐ or muscle‐specific depletion of CLPP. However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold‐induced stress due to non‐functional adaptive thermogenesis. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high‐fat diet feeding and cold exposure providing tools to understand pathologies with deregulated Clpp expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.     

“Pulling the plug” on cellular copper: The role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

"Pulling the plug" on cellular copper: the role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

Transporters for amino acids in plant cells: some functions and many unknowns

Membrane proteins are essential to move amino acids in or out of plant cells as well as between organelles. While many putative amino acid transporters have been identified, function in nitrogen movement in plants has only been shown for a few proteins. Those studies demonstrate that import systems are fundamental in partitioning of amino acids at cellular and whole plant level. Physiological data further suggest that amino acid transporters are key-regulators in plant metabolism and that their activities affect growth and development. By contrast, knowledge on the molecular mechanisms of cellular export processes as well as on intracellular transport of amino acids is scarce. Similarly, little is known about the regulation of amino acid transporter function and involvement of the transporters in amino acid signaling. Future studies need to identify the missing components to elucidate the importance of amino acid transport processes for whole plant physiology and productivity.     

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.     

Alteration of Mitochondrial Proteome Due to Activation of Notch1 Signaling Pathway

The Notch signaling pathway, a known regulator of cell fate decisions, proliferation, and apoptosis, has recently been implicated in the regulation of glycolysis, which affects tumor progression. However, the impact of Notch on other metabolic pathways remains to be elucidated. To gain more insights into the Notch signaling and its role in regulation of metabolism, we studied the mitochondrial proteome in Notch1-activated K562 cells using a comparative proteomics approach. The proteomic study led to the identification of 10 unique proteins that were altered due to Notch1 activation. Eight of these proteins belonged to mitochondria-localized metabolic pathways like oxidative phosphorylation, glutamine metabolism, Krebs cycle, and fatty acid oxidation. Validation of some of these findings showed that constitutive activation of Notch1 deregulated glutamine metabolism and Complex 1 of the respiratory chain. Furthermore, the deregulation of glutamine metabolism involved the canonical Notch signaling and its downstream effectors. The study also reports the effect of Notch signaling on mitochondrial function and status of high energy intermediates ATP, NADH, and NADPH. Thus our study shows the effect of Notch signaling on mitochondrial proteome, which in turn affects the functioning of key metabolic pathways, thereby connecting an important signaling pathway to the regulation of cellular metabolism.     

生理病理状态下线粒体合成ATP动态调控机制

线粒体是细胞的代谢中心之一，不仅产生大量的ATP为细胞提供能量，还参与多种生物分子（例如核酸、氨基酸、胆固醇和脂肪酸）合成及代谢废物的处理。ATP是细胞重要的“能源货币”，是能量载体和信号分子，参与调节细胞的各种生命活动。动物与人在激烈运动时，ATP消耗速率增加数十倍，但细胞内的ATP仍维持在“设定点”水平，不出现降低。因此，传统生理学观点认为，动物细胞内ATP水平保持恒定。但新的研究结果表明，生物细胞内ATP水平存在波动。生理条件下，增加能量物资（糖、脂和氨基酸等）和氧供，促进线粒体ATP合成，可使细胞内ATP水平出现一过性升高。新的研究证明，在肥胖情况下，由于能量物质的过多供应，细胞内ATP水平出现持续性升高,构成代谢紊乱的源头信号。线粒体ATP合成受多种因素影响，如氧化应激、钙超载、缺氧、线粒体膜通透性增加和线粒体DNA突变等。这些因素与疾病条件下细胞内ATP水平持续降低相关，常见的疾病包括阿尔茨海默症、帕金森疾病、精神分裂症、肿瘤、心衰、全身炎症反应综合征等。本综述简要概述线粒体调节细胞内ATP水平的研究进展，重点讨论造成ATP波动的因素、机制及病理生理学意义。     

Kinase signaling cascades in the mitochondrion: a matter of life or death

In addition to powering energy needs of the cell, mitochondria function as pivotal integrators of cell survival/death signals. In recent years, numerous studies indicate that each of the major kinase signaling pathways can be stimulated to target the mitochondrion. These include protein kinase A, protein kinase B/Akt, protein kinase C, extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. Although most studies focus on phosphorylation of pro- and antiapoptotic proteins (BAD, Bax, Bcl-2, Bcl-xL), kinase-mediated regulation of complex I activity, anion and cation channels, metabolic enzymes, and Mn-SOD mRNA has also been reported. Recent identification of a number of scaffold proteins (AKAP, PICK, Sab) that bring specific kinases to the cytoplasmic surface of mitochondria further emphasizes the importance of mitochondrial kinase signaling. Immunogold electron microscopy, subcellular fractionation and immunofluorescence studies demonstrate the presence of kinases within subcompartments of the mitochondrion, following diverse stimuli and in neurodegenerative diseases. Given the sensitivity of these signaling pathways to reactive oxygen and nitrogen species, in situ activation of mitochondrial kinases may represent a potent reverse-signaling mechanism for communication of mitochondrial status to the rest of the cell.     

生理病理状态下线粒体合成ATP动态调控机制

线粒体是细胞的代谢中心之一,不仅产生大量的ATP为细胞提供能量,还参与多种生物分子（例如核酸、氨基酸、胆固醇和脂肪酸）合成及代谢废物的处理。ATP是细胞重要的“能源货币”,是能量载体和信号分子,参与调节细胞的各种生命活动。动物与人在激烈运动时,ATP消耗速率增加数十倍,但细胞内的ATP仍维持在“设定点”水平,不出现降低。因此,传统生理学观点认为,动物细胞内ATP水平保持恒定。但新的研究结果表明,生物细胞内ATP水平存在波动。生理条件下,增加能量物资（糖、脂和氨基酸等）和氧供,促进线粒体ATP合成,可使细胞内ATP水平出现一过性升高。新的研究证明,在肥胖情况下,由于能量物质的过多供应,细胞内ATP水平出现持续性升高,构成代谢紊乱的源头信号。线粒体ATP合成受多种因素影响,如氧化应激、钙超载、缺氧、线粒体膜通透性增加和线粒体DNA突变等。这些因素与疾病条件下细胞内ATP水平持续降低相关,常见的疾病包括阿尔茨海默症、帕金森疾病、精神分裂症、肿瘤、心衰、全身炎症反应综合征等。本综述简要概述线粒体调节细胞内ATP水平的研究进展,重点讨论造成ATP波动的因素、机制及病理生理学意义。     

Control of mitochondrial integrity in ageing and disease

Various molecular and cellular pathways are active in eukaryotes to control the quality and integrity of mitochondria. These pathways are involved in keeping a ‘healthy’ population of this essential organelle during the lifetime of the organism. Quality control (QC) systems counteract processes that lead to organellar dysfunction manifesting as degenerative diseases and ageing. We discuss disease- and ageing-related pathways involved in mitochondrial QC: mtDNA repair and reorganization, regeneration of oxidized amino acids, refolding and degradation of severely damaged proteins, degradation of whole mitochondria by mitophagy and finally programmed cell death. The control of the integrity of mtDNA and regulation of its expression is essential to remodel single proteins as well as mitochondrial complexes that determine mitochondrial functions. The redundancy of components, such as proteases, and the hierarchies of the QC raise questions about crosstalk between systems and their precise regulation. The understanding of the underlying mechanisms on the genomic, proteomic, organellar and cellular levels holds the key for the development of interventions for mitochondrial dysfunctions, degenerative processes, ageing and age-related diseases resulting from impairments of mitochondria.     

Control of cell metabolism by the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) triggers the activation of many intracellular signals that control cell proliferation, growth, survival, migration, and differentiation. Given its wide expression, EGFR has many functions in development and tissue homeostasis. Some of the cellular outcomes of EGFR signaling involve alterations of specific aspects of cellular metabolism, and alterations of cell metabolism are emerging as driving influences in many physiological and pathophysiological contexts. Here we review the mechanisms by which EGFR regulates cell metabolism, including by modulation of gene expression and protein function leading to control of glucose uptake, glycolysis, biosynthetic pathways branching from glucose metabolism, amino acid metabolism, lipogenesis, and mitochondrial function. We further examine how this regulation of cell metabolism by EGFR may contribute to cell proliferation and differentiation and how EGFR-driven control of metabolism can impact certain diseases and therapy outcomes.     

Metabolic regulation by SIRT3: implications for tumorigenesis

Cancer cells meet their needs for energy and biomass production by consuming high levels of nutrients and rewiring metabolism to support macromolecular biosynthesis. Mitochondrial enzymes play central roles in anabolic growth, and acetylation may provide a key layer of regulation over mitochondrial metabolic pathways. As a major mitochondrial deacetylase, SIRT3 regulates the activity of enzymes to coordinate global shifts in cellular metabolism. SIRT3 promotes the function of the tricarboxylic acid (TCA) cycle and the electron transport chain and reduces oxidative stress. Loss of SIRT3 triggers oxidative damage, reactive oxygen species (ROS)-mediated signaling, and metabolic reprogramming to support proliferation and tumorigenesis. Thus, SIRT3 is an intriguing example of how nutrient-sensitive, post-translational regulation may provide integrated regulation of metabolic pathways to promote metabolic homeostasis in response to diverse nutrient signals.     

Regulatory analysis of amino acid synthesis pathway in Escherichia coli: aspartate family

Proteins have various compositions of twenty specific naturally occurring amino acids. In spite of their importance in cellular metabolism, biosynthesis mechanisms, changing control conditions, and affection of effectors are not clearly understood yet. So we have made an effort to elucidate the details of metabolic control mechanisms in amino acid synthesis pathways through examining an extensive database search. In this study, we have newly constructed six amino acid biosynthesis pathways including aspartate, asparagine, methionine, threonine, isoleucine, and lysine, which we call the aspartate family. They contain the major reaction mechanisms, which inhibitory control loops and activating compounds. Moreover, we have tried to collect all of the effectors which might affect the aspartate family biosynthetic networks.     

Overexpression of UCP-3 in skeletal muscle of mice results in increased expression of mitochondrial thioesterase mRNA

Mice overexpressing human UCP-3 in skeletal muscle (UCP-3tg) are lean despite overeating, have increased metabolic rate, and their skeletal muscle mitochondria show increased proton conductance. The true function of UCP-3 however, has yet to be determined. It is assumed that UCP-3tg mice have increased fatty acid beta-oxidation to fuel their increased metabolic rate. In this study we have quantified skeletal muscle mRNA levels of a number of genes involved in fatty acid metabolism. mRNA levels of uncoupling protein-2, carnitine palmitoyl transferase-1beta and fatty acid binding proteins, and transporters were unchanged when compared to wild-type mice. Lipoprotein lipase mRNA was slightly, but significantly, increased by 50%. The most notable change in gene expression was a threefold increase in mitochondrial thioesterase (MTE-1) expression. In the face of a chronic increase in mitochondrial uncoupling these changes suggest that increased flux of fatty acids through the beta-oxidation pathway does not necessarily require marked changes in expression of genes involved in fatty acid metabolism. The large increase in MTE-1 both confirms the importance of this gene in situations where mitochondrial beta-oxidation is increased and supports the hypothesis that UCP-3 exports fatty acids generated by MTE-1 in the mitochondrion.