Metabolic flux and the regulation of mammalian cell growth

The study of normal mammalian cell growth and the defects that contribute to disease pathogenesis links metabolism to cell growth. Here, we visit several aspects of growth-promoting metabolism, emphasizing recent advances in our understanding of how alterations in glucose metabolism affect cytosolic and mitochondrial redox potential and ATP generation. These alterations drive cell proliferation not only through supporting biosynthesis, energy metabolism, and maintaining redox potential but also through initiating signaling mechanisms that are still poorly characterized. The evolutionary basis of these additional layers of growth control is also discussed.     

Control theory of metabolic channelling

Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control.This chapter develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy form the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives methods functionally to diagnose the extent to which channelling and metabolite sequestration occur.     

综述: 细胞自噬和线粒体质量控制与健康衰老

拥有健康的晚年是每一个人的祈盼,这也是目前应对即将到来的社会老龄化危机而需要解决的重要课题.实现健康衰老需要对人类衰老发生的机制有深入的了解,比如在此过程中扮演着重要角色的线粒体的研究.线粒体是细胞能量和自由基代谢中心,也是细胞凋亡调控中心,并在信号转导和基因表达调控中发挥重要作用.线粒体一旦受损,一方面能量代谢发生紊乱,另一方面产生大量自由基,影响细胞的正常生长,并导致细胞甚至机体的衰老.正常情况下,细胞通过自噬溶酶体机制选择性清除受损伤和不需要的线粒体,这是线粒体质量控制的重要机制.研究发现,线粒体质量控制异常可能在衰老发生过程中起关键作用.限食及增强运动能有效促进线粒体质量控制,改善线粒体功能并延缓衰老.     

Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells

Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.     

The role of mitochondria in the life of the nematode,Caenorhabditis elegans

Mitochondria are essential organelles involved in energy metabolism via oxidative phosphorylation. They play a vital role in diverse biological processes such as aging and apoptosis. In humans, defects in the mitochondrial respiratory chain (MRC) are responsible for or associated with a bewildering variety of diseases. The nematode Caenorhabditis elegans is a simple animal and a powerful genetic and developmental model system. In this review, we discuss how the nematode model system has contributed to our understanding of mitochondrial dynamics, of the genetics and inheritance of the mitochondrial genome, and of the consequences of nuclear and mitochondrial DNA (mtDNA) mutations. Mitochondrial respiration is vital to energy metabolism but also to other aspects of multicellular life such as aging and development. We anticipate that further significant contributions to our understanding of mitochondrial function in animal biology are forthcoming with the C. elegans model system.     

The Phosphatase Ptc7 Induces Coenzyme Q Biosynthesis by Activating the Hydroxylase Coq7 in Yeast

The study of the components of mitochondrial metabolism has potential benefits for health span and lifespan because the maintenance of efficient mitochondrial function and antioxidant capacity is associated with improved health and survival. In yeast, mitochondrial function requires the tight control of several metabolic processes such as coenzyme Q biosynthesis, assuring an appropriate energy supply and antioxidant functions. Many mitochondrial processes are regulated by phosphorylation cycles mediated by protein kinases and phosphatases. In this study, we determined that the mitochondrial phosphatase Ptc7p, a Ser/Thr phosphatase, was required to regulate coenzyme Q₆ biosynthesis, which in turn activated aerobic metabolism and enhanced oxidative stress resistance. We showed that Ptc7p phosphatase specifically activated coenzyme Q₆ biosynthesis through the dephosphorylation of the demethoxy-Q₆ hydroxylase Coq7p. The current findings revealed that Ptc7p is a regulator of mitochondrial metabolism that is essential to maintain proper function of the mitochondria by regulating energy metabolism and oxidative stress resistance.     

Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans- membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria- associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.     

Control theory of metabolic channelling

Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control.This paper develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy from the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives methods functionally to diagnose the extent to which channelling and metabolite sequestration occur.This article was published in Molecular and Cellular Biochemistry133/134: 313–331, 1994. Kluwer Academic Publishers regret the publication of the uncorrected version.     

Hypoxia affects mitochondrial protein expression in rat skeletal muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500?m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.     

Internal regulation of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

Previously [Ainscow, E.K. & Brand, M.D. (1999) Eur. J. Biochem. 263, 671-685], top-down control analysis was used to describe the control pattern of energy metabolism in rat hepatocytes. The system was divided into nine reaction blocks (glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption) linked by five intermediates (intracellular glucose 6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential). The kinetic responses (elasticities) of reaction blocks to intermediates were determined and used to calculate control coefficients. In the present paper, these elasticities and control coefficients are used to quantify the internal regulatory pathways within the cell. Flux control coefficients were partitioned to give partial flux control coefficients. These describe how strongly one block of reactions controls the flux through another via its effects on the concentration of a particular intermediate. Most flux control coefficients were the sum of positive and negative partial effects acting through different intermediates; these partial effects could be large compared to the final control strength. An important result was the breakdown of the way ATP consumption controlled respiration: changes in ATP level were more important than changes in mitochondrial membrane potential in stimulating oxygen consumption when ATP consumption increased. The partial internal response coefficients to changes in each intermediate were also calculated; they describe how steady state concentrations of intermediates are maintained. Increases in mitochondrial membrane potential were opposed mostly by decreased supply, whereas increases in glucose-6-phosphate, NADH/NAD and pyruvate were opposed mostly by increased consumption. Increases in ATP were opposed significantly by both decreased supply and increased consumption.     

Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.     

Down-regulated energy metabolism genes associated with mitochondria oxidative phosphorylation and fatty acid metabolism in viral cardiomyopathy mouse heart

The majority of experimental and clinical studies indicates that the hypertrophied and failing myocardium are characterized by changes in energy and substrate metabolism that attributed to failing heart changes at the genomic level, in fact, heart failure is caused by various diseases, their energy metabolism and substrate are in different genetic variations, then the potential significance of the molecular mechanisms for the aetiology of heart failure is necessary to be evaluated. Persistent viral infection (especially coxsackievirus group B3) of the myocardium in viral myocarditis and viral dilated cardiomyopathy has never been neglected by experts. This study aimed to explore the role and regulatory mechanism of the altered gene expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism in viral dilated cardiomyopathy. cDNA Microarray technology was used to evaluate the expression of >35,852 genes in a mice model of viral dilated cardiomyopathy. In total 1385 highly different genes expression, we analyzed 33 altered genes expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and further selected real-time-PCR for quantity one of regulatory mechanisms for energy including fatty acid metabolism—the UCP2 and assayed cytochrome C oxidase activity by Spectrophotometer to explore mitochondrial oxidative phosphorylation function. We found obviously different expression of 33 energy metabolism genes associated with mitochondria oxidative phosphorylation, fatty acid metabolism in cardiomyopathy mouse heart, the regulatory gene for energy metabolism: UCP2 was down-regulated and cytochrome C oxidase activity was decreased. Genes involved in both fatty acid metabolism and mitochondrial oxidative phosphorylation were down-regulated, mitochondrial uncoupling proteins (UCP2) expression did not increase but decrease which might be a kind of adaptive protection response to regulate energy metabolism for ATP produce.     

The Role of Mitochondrial Quality Control in Cognitive Dysfunction in Diabetes

Type 2 diabetes (T2DM) is a well known risk factor for Alzheimer’s disease. Mitochondria are the center of intracellular energy metabolism and the main source of reactive oxygen species. Mitochondrial dysfunction has been identified as a key factor in diabetes-associated brain alterations contributing to neurodegenerative events. Defective insulin signaling may act in concert with neurodegenerative mechanisms leading to abnormalities in mitochondrial structure and function. Mitochondrial dysfunction triggers neuronal energy exhaustion and oxidative stress, leading to brain neuronal damage and cognitive impairment. The normality of mitochondrial function is basically maintained by mitochondrial quality control mechanisms. In T2DM, defects in the mitochondrial quality control pathway in the brain have been found to lead to mitochondrial dysfunction and cognitive impairment. Here, we discuss the association of mitochondrial dysfunction with T2DM and cognitive impairment. We also review the molecular mechanisms of mitochondrial quality control and impacts of mitochondrial quality control on the progression of cognitive impairment in T2DM.

     

Evaluating the roles of energetic functional constraints on teleost mitochondrial-encoded protein evolution

Mitochondria are the power plant of cells, which play critical roles not only in energy metabolism but also in thermoregulation. These two roles have been individually suggested to influence mitochondrial DNA (mtDNA) evolution, however their relative importance is still rarely considered. Here, we conduct a comparative genomic analysis of 401 teleost complete mitochondrial genomes and test the roles of these dual functional constraints on mitochondria to provide a more complete view of mtDNA evolution. We found that mitochondrial protein-coding genes of migratory fishes have significantly smaller Ka/Ks than nonmigratory fishes. The same data set showed that the genes of fishes living in cold climates have significantly smaller Ka/Ks than tropical fishes. In contrast, these trends were not observed for two nuclear genes that are not involved in energy metabolism. The differences in selection patterns observed between mitochondrial and nuclear genes suggest that the functional constraints acting on mitochondria, due to energy metabolism and/or thermoregulation, influence the evolution of mitochondrial-encoded proteins in teleosts.     

Mitochondria in cancer cells: what is so special about them?

The past decade has revealed a new role for the mitochondria in cell metabolism--regulation of cell death pathways. Considering that most tumor cells are resistant to apoptosis, one might question whether such resistance is related to the particular properties of mitochondria in cancer cells that are distinct from those of mitochondria in non-malignant cells. This scenario was originally suggested by Otto Warburg, who put forward the hypothesis that a decrease in mitochondrial energy metabolism might lead to development of cancer. This review is devoted to the analysis of mitochondrial function in cancer cells, including the mechanisms underlying the upregulation of glycolysis, and how intervention with cellular bioenergetic pathways might make tumor cells more susceptible to anticancer treatment and induction of apoptosis.     

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage‐directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.     

Mitochondrial sirtuins,metabolism, and aging

Maintaining metabolic homeostasis is essential for cellular and organismal health throughout life. Multiple signaling pathways that regulate metabolism also play critical roles in aging, such as PI3K/AKT, mTOR, AMPK, and sirtuins (SIRTs). Among them, sirtuins are known as a protein family with versatile functions, such as metabolic control, epigenetic modification and lifespan extension. Therefore, by understanding how sirtuins regulate metabolic processes, we can start to understand how they slow down or accelerate biological aging from the perspectives of metabolic regulation. Here, we review the biology of SIRT3, SIRT4, and SIRT5, known as the mitochondrial sirtuins due to their localization in the mitochondrial matrix. First, we will discuss canonical pathways that regulate metabolism more broadly and how these are integrated with aging regulation. Then, we will summarize the current knowledge about functional differences between SIRT3, SIRT4, and SIRT5 in metabolic control and integration in signaling networks. Finally, we will discuss how mitochondrial sirtuins regulate processes associated with aging and aging-related diseases.