Astrocytes and energy metabolism

Astrocytes are glial cells, which play a significant role in a number of processes, including the brain energy metabolism. Their anatomical position between blood vessels and neurons make them an interface for effective glucose uptake from blood. After entering astrocytes, glucose can be involved in different metabolic pathways, e.g. in glycogen production. Glycogen in the brain is localized mainly in astrocytes and is an important energy source in hypoxic conditions and normal brain functioning. The portion of glucose metabolized into glycogen molecules in astrocytes is as high as 40%. It is thought that the release of gliotransmitters (such as glutamate, neuroactive peptides and ATP) into the extracellular space by regulated exocytosis supports a significant part of communication between astrocytes and neurons. On the other hand, neurotransmitter action on astrocytes has a significant role in brain energy metabolism. Therefore, understanding the astrocytes energy metabolism may help understanding neuron-astrocyte interactions.     

AMPK: a nutrient and energy sensor that maintains energy homeostasis

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.     

Relation of actin fibrils to energy metabolism of endothelial cells

Summary The physiological significance of the association of glycolytic enzymes with actin fibrils was investigated in cell culture. Cytochalasin D (CD) was used to induce the known actin-based sequence of events in a culture of an endothelial-cell line (XTH-2) derived from hearts from tadpoles of Xenopus laevis. 1 min following addition of CD, ruptures in the cortical fibrillar meshwork and in stress fibres are seen. At the same time the cellular ATP level decreases by ca. 25%. This and the following reactions resulting in a kind of arborization depend on a continuous supply with metabolic energy. As shown by measurements of oxygen consumption, cells with intact energy metabolism provide the ATP needed from glycolysis; ATP produced by oxidative phosphorylation is not ultilized as long as lactate dehydrogenase (LDH) reoxidizes NADH₂. After inhibition of LDH, respiration in XTH-2 cells doubles. CD treatment induces a transient increase in oxygen consumption, indicating an increased energy supply by respiration. From these results we conclude: The energy needed by the actomyosin system is — under normal metabolic conditions — supplied from ATP phosphorylated in glycolysis. The processes of energy metabolism seem to be highly compartmentalized; ATP is not a parameter that is kept constant in time intervals of minutes up to one hour.     

Stabilization of energy charge, generation of oscillations and multiple steady states in energy metabolism as a result of purely stoichiometric regulation.

A simple kinetic model of cell energy metabolism with autocatalytic reaction sequences has beepn analysed. The model accounts for the fact that part of energy produced in the form of ATP, or any other equivalent form, is utilized in "sparking" reactions to activate initial substrates. Analysis of the model shows that energy metabolism, in the absen-e of all non-stoichiometric (i.e. isosteric, cooperative, and allosteric) regulations, is capable of (a) stabilizing, to a high degree of accuracy, the relative concentration of the "charged form" of the energy-transferring cofactor (ATP); (b) alternating between two stable stationary states by means of hysteretic transitions; (c) generating self-oscillations in energy production. It is proposed that energy metabolism can be a source of very slow, in particular circadian (of about a one-day period), oscillations which may serve as the basis for temporal organization of the cell.     

N-acetyl-p-benzoquinone imine-induced changes in the energy metabolism in hepatocytes

The effect of N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, on the energy metabolism in isolated hepatocytes was investigated. Incubation of cells with NAPQI (400 microM) resulted in an immediate uptake into the mitochondria, followed by both reduction and glutathione conjugation of the quinone imine. These reactions were extremely rapid and were associated with depletion of the mitochondrial ATP content (greater than 80% depletion after 1 min exposure). The loss of ATP was accompanied by increases in ADP and AMP, as well as NADP. No effect on mitochondrial NAD was observed during this initial phase. Similar alterations were produced by NAPQI in the cytosolic compartment. Furthermore, incubation of hepatocytes with NAPQI inhibited oxygen consumption by nearly 90% within 10 s. In parallel to these biochemical changes, there was marked bleb formation on the surface of the hepatocytes, which was found to precede cell death (trypan blue uptake). In conclusion, our results demonstrate that during exposure of hepatocytes to NAPQI, dramatic changes in cellular energy metabolism occur. These biochemical alterations may be caused by a rapid decrease in mitochondrial function, and they may play an important role in the initiation of NAPQI-induced cytotoxicity.     

Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: modeling studies of stationary and oscillatory states

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S(1)), in the presence of a second substrate, formate (S(2)), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S(2) allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S(1) which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S(1)) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (eta) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S(2)/S(1) ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of eta was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high eta predicting that higher biomass yields may be obtained through a variable, high, eta by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.     

Activation of an energy providing response in human keratinocytes after gamma irradiation

We performed a microarray study on human differentiated HaCaT keratinocytes exposed to ionizing radiation (2 or 10 Gy). At 3 h after exposure, more than 150 known and unknown genes were found regulated in irradiated HaCaT keratinocytes. Among the genes regulated at 3 h, those involved in cell energy metabolism appeared to be the most abundant and the most responsive. Two mitochondrial ATP-synthases and several other genes involved in energy producing pathways, such as glucose metabolism, were induced, whereas many genes from energy requiring pathways were shut down. These changes in energy metabolism were confirmed both in normal primary keratinocytes and in HaCaT keratinocytes by RT-PCR and proteins studies. Moreover, measures of intracellular ATP revealed a 50% increase in keratinocytes immediately after irradiation, supporting an energy procurement response. The overall results indicate that irradiation induces an immediate burst of ATP that seems to be a general response of human differentiated keratinocytes to the radiation stress. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/v95.html     

Short-term inhibition of the energy metabolism affects motility but not surface properties of sperm cells

Centrifugal countercurrent distribution (CCCD) in aqueous two-phase systems has been proven to be a useful method to study subtle surface properties of spermatozoa. The present work shows that a short-term inhibition of the energy metabolism of sperm cells effected by incubating bovine sperm cells with KCN or ouabain, did not account for changes in the cell surface properties, as assessed either by estimation of the cell viability or by CCCD analysis. However, the short-term inhibition of energy metabolism provoked a clear decrease of cell motility, suggesting that a drop of cellular ATP levels brings about a rapid decrease of motility followed by a very delayed effect on cell surface properties. The relevance of these results on the handling of sperm and on the understanding of the molecular events underlying asthenospermia is discussed.     

The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon

The microbiome is being characterized by large-scale sequencing efforts, yet it is not known whether it regulates host metabolism in a general versus tissue-specific manner or which bacterial metabolites are important. Here, we demonstrate that microbiota have a strong effect on energy homeostasis in the colon compared to other tissues. This tissue specificity is due to colonocytes utilizing bacterially produced butyrate as their primary energy source. Colonocytes from germfree mice are in an energy-deprived state and exhibit decreased expression of enzymes that catalyze key steps in intermediary metabolism including the TCA cycle. Consequently, there is a marked decrease in NADH/NAD(+), oxidative phosphorylation, and ATP levels, which results in AMPK activation, p27(kip1) phosphorylation, and autophagy. When butyrate is added to germfree colonocytes, it rescues their deficit in mitochondrial respiration and prevents them from undergoing autophagy. The mechanism is due to butyrate acting as an energy source rather than as an HDAC inhibitor.     

Continuous cell-free protein synthesis using glycolytic intermediates as energy sources

In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis more realistic alternative for rapid protein production.     

Reinstatement of the ATP high energy paradigm

The paradigm that the hydrolysis of ATP releases high Gibbs energy able to perform work has increasingly been questioned over the last two decades. Results from theoretical and experimental studies have been interpreted to indicate that the synthesis of ATP from ADP and P_i does not require energy supply and that binding of ATP per se can transmit utilizable energy to an enzyme. As has recently been concluded, all this has led to a change of the ATP high energy paradigm in bioenergetics. Starting from this challenge, the present review singles out the striking sources of the apparent dichotomy in biocnergetics, and endeavours to eliminate the apparent contradictions by the application of the prior knowledge on both the participation of the enzyme protein in energy exchange processes and the particular reactivities of phosphorus that make it an outstanding element for functionally variable work assignments in enzymatic systems.     

Astrocyte activation in working brain: energy supplied by minor substrates

Glucose delivered to brain by the cerebral circulation is the major and obligatory fuel for all brain cells, and assays of functional activity in working brain routinely focus on glucose utilization. However, these assays do not take into account the contributions of minor substrates or endogenous fuel consumed by astrocytes during brain activation, and emerging evidence suggests that glycogen, acetate, and, perhaps, glutamate, are metabolized by working astrocytes in vivo to provide physiologically significant amounts of energy in addition to that derived from glucose. Rates of glycogenolysis during sensory stimulation of normal, conscious rats are high enough to support the notion that glycogen can contribute substantially to astrocytic glucose utilization during activation. Oxidative metabolism of glucose provides most of the ATP for cultured astrocytes, and a substantial contribution of respiration to astrocyte energetics is supported by recent in vivo studies. Astrocytes preferentially oxidize acetate taken up into brain from blood, and calculated local rates of acetate utilization in vivo are within the range of calculated rates of glucose oxidation in astrocytes. Glutamate may also serve as an energy source for activated astrocytes in vivo because astrocytes in tissue culture and in adult brain tissue readily oxidize glutamate. Taken together, contributions of minor metabolites derived from endogenous and exogenous sources add substantially to the energy obtained by astrocytes from blood-borne glucose. Because energy-generating reactions from minor substrates are not taken into account by routine assays of functional metabolism, they reflect a "hidden cost" of astrocyte work in vivo.     

Implication of liver cardiolipins in mitochondrial energy metabolism disorder in cancer cachexia

Mitochondrial membranes are essential for the good functioning of the organelle. For instance, the inner mitochondrial membrane contains the oxidative phosphorylation system that permits ATP synthesis. Phospholipids environment and especially cardiolipin are crucial for the mitochondrial energy metabolism. Indeed, cardiolipin is known to provide essential structural and functional support to several proteins involved in oxidative phosphorylation. Alterations in cardiolipin structure, content and fatty acids composition have been associated with mitochondrial dysfunction in several physiopathological conditions and diseases. Cancer cachexia is a complex and dynamic process characterized by a negative energy balance induced by anorexia and hypermetabolism which leads to a drastic loss in body weight that aggravate prognosis of cancer patients. The underlying mechanisms of hypermetabolism are not fully understood. Whether the mitochondrial energy metabolism is altered during this disease and may participate to hypermetabolism is not clear. This mini-review focuses on cardiolipin especially its biosynthesis and remodeling pathways, its relation with mitochondrial energy metabolism and its possible implication in the cancer cachexia syndrome.     

Structural bioenergetics and energy transduction mechanisms.

Life depends on transduction processes that couple cellular metabolism to environmental energy sources such as light or reduced compounds. These primary energy sources must be efficiently converted into forms that can be utilized by cells for biosynthesis, motility, transport, regulation, and other metabolic functions. In recent years, there has been an explosive increase in the determination of structures for proteins mediating energy transduction processes. These developments provide the opportunity to evaluate the structural basis for the efficient coupling of two energetic processes, which defines the area of structural bioenergetics. Here, we present some general features of energy transduction processes, including arguments that effective coupling of two processes by a transduction protein occurs by way of conformational states that are common to the catalysis of each process. This is illustrated by examples from the nucleotide switch family of proteins, with emphasis on the nitrogenase system where ATP hydrolysis is coupled to an electron transfer reaction.     

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.     

In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.     

Amoeboid movement in human leucocytes: basic mechanisms, cytobiological and clinical significance.

The present paper is an analytical review of the information available on amoeboid movement in human leucocytes. The reported evidence suggests that leucocyte locomotion is due to pressure developed in the cell cortex in the middle and posterior parts of the moving cell, that 4 nm fibrils may provide at least part of the ultrastructural basis of locomotion, that actin-like and myosin-like proteins may be involved in the mechanism of movement and that ATP may serve as an energy source. Leucocyte motility appears to be governed mainly by factors produced in the external medium. Neutrophil chemotaxis is the most antitubulin-susceptible cell mechanism known; from this observation an essential role of microtubule redistribution in chemotaxis is inferred. In contrast, the random movement of neutrophils is not appreciably affected by antimitotic concentrations of antitubulins. Amoeboid movement seems to be an important mechanism in the short-distance locomotion and immunological functions of leucocytes.     

Trimethylamine and trimethylamine N-oxide are supplementary energy sources for a marine heterotrophic bacterium: implications for marine carbon and nitrogen cycling

Bacteria of the marine Roseobacter clade are characterised by their ability to utilise a wide range of organic and inorganic compounds to support growth. Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are methylated amines (MA) and form part of the dissolved organic nitrogen pool, the second largest source of nitrogen after N₂ gas, in the oceans. We investigated if the marine heterotrophic bacterium, Ruegeria pomeroyi DSS-3, could utilise TMA and TMAO as a supplementary energy source and whether this trait had any beneficial effect on growth. In R. pomeroyi, catabolism of TMA and TMAO resulted in the production of intracellular ATP which in turn helped to enhance growth rate and growth yield as well as enhancing cell survival during prolonged energy starvation. Furthermore, the simultaneous use of two different exogenous energy sources led to a greater enhancement of chemoorganoheterotrophic growth. The use of TMA and TMAO primarily as an energy source resulted in the remineralisation of nitrogen in the form of ammonium, which could cross feed into another bacterium. This study provides greater insight into the microbial metabolism of MAs in the marine environment and how it may affect both nutrient flow within marine surface waters and the flux of these climatically important compounds into the atmosphere.     

Effects of altering carbohydrate metabolism on energy status and contractile function of vascular smooth muscle

Substrate-dependent changes in vascular smooth muscle energy metabolism and contractile function were investigated in isolated porcine carotid arteries. In media containing glucose glycogen catabolism accounted for all the estimated high-energy phosphate turnover that occurred in conjunction with contraction induced by 80 mM KCl. However, in glucose-free media glycogen catabolism accounted for only a portion of the estimated ATP utilization in resting and contracting arteries, even though glycogen stores were not depleted. The glycogenolysis and lactate production that ordinarily accompanies contraction was completely inhibited by 5 mM 2-deoxyglucose (2-DG). However, there was no decrease in the high-energy phosphate levels when compared to control resting arteries similarly treated with 2-DG. The results suggest that an endogenous non-carbohydrate source may be an important substrate for energy metabolism. Treatment of arteries with 50 microM iodoacetate (IA) in media containing glucose resulted in a marked reduction of high energy phosphate levels and an accumulation of phosphorylated glycolytic intermediates, as demonstrated by 31P-NMR spectroscopy. In glucose-free media, 50 microM IA had only a slight effect on high-energy phosphate levels, while glycogenolysis proceeded unhindered. With 1 mM IA in glucose-free media, the oxidative metabolism of glycogen was inhibited as evidenced by the depletion of high-energy phosphates and the appearance of sugar phosphates in the 31P-NMR spectra. Thus, the titration of enzyme systems with IA reveals a structural partitioning of carbohydrate metabolism, as suggested by previous studies.