NAMPT-mediated salvage synthesis of NAD+ controls morphofunctional changes of macrophages.

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+'s cytosolic role in the regulation of morphofunctional characteristics of macrophages.

Glucose controls morphodynamics of LPS-stimulated macrophages.

Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics.

Complete brain-type creatine kinase deficiency in mice blocks seizure activity and affects intracellular calcium kinetics.

PURPOSE: Brain-type creatine kinase (CK-B) and ubiquitous mitochondrial creatine kinase (UbCKmit) act as components of local phosphocreatine ATP shuttles that help in the compartmentalization and maintenance of pools of high-energy phosphate molecules in both neurons and glial cells. We investigated the role of these brain-type creatine kinases during extreme energy-demanding conditions in vivo (generalized tonic-clonic seizures) and in vitro. METHODS: The physiologic response of wild-types and mice lacking both CK-B and UbCKmit (CK--/--mice) to pentylenetetrazole (PTZ)-induced seizures was measured using electroencephalography (EEG) recordings and behavioral monitoring. In vitro intracellular Ca(2+) kinetics in hippocampal granule neurons were monitored upon single and repetitive depolarizations. RESULTS: PTZ induced in only a few CK--/-- mice PTZ seizure-like behavior, but in all wild-types a full-blown seizure. EEG analysis showed that preseizure jerking was associated with high-amplitude discharges. Wild-type EEG recordings showed continuous runs of rhythmic 4-6 Hz activity, whereas no rhythmic EEG activities were observed in the few CK--/-- mice that developed a behavioral seizure. All other CK--/-- mice displayed a sudden postictal depression without any development of a generalized seizure. Hippocampal granule neurons of CK--/-- mice displayed a higher Ca(2+) removal speed following repetitive KCl-induced depolarizations. DISCUSSION: Deficiency for creatine kinase is affecting brain energy metabolism and will likely contribute to the disturbance of seizure development. Because CK--/-- hippocampal neurons exhibited an increase in Ca(2+) removal rate of elevated intracellular levels, we conclude that altered Ca(2+) clearance in CK--/-- neurons could play a role in the abnormal EEG and seizure activity.

Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype.

BACKGROUND: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. RESULTS: Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. CONCLUSION: The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

Mice lacking brain-type creatine kinase activity show defective thermoregulation.

The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0 degrees C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK--/-- mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK--/-- mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect.

Local ATP generation by brain-type creatine kinase (CK-B) facilitates cell motility.

BACKGROUND: Creatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. CONCLUSION/SIGNIFICANCE: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption.

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Creatine kinase B deficient neurons exhibit an increased fraction of motile mitochondria.

BACKGROUND: Neurons require an elaborate system of intracellular transport to distribute cargo throughout axonal and dendritic projections. Active anterograde and retrograde transport of mitochondria serves in local energy distribution, but at the same time also requires input of ATP. Here we studied whether brain-type creatine kinase (CK-B), a key enzyme for high-energy phosphoryl transfer between ATP and CrP in brain, has an intermediary role in the reciprocal coordination between mitochondrial motility and energy distribution. Therefore, we analysed the impact of brain-type creatine kinase (CK-B) deficiency on transport activity and velocity of mitochondria in primary murine neurons and made a comparison to the fate of amyloid precursor protein (APP) cargo in these cells, using live cell imaging. RESULTS: Comparison of average and maximum transport velocities and global transport activity showed that CK-B deficiency had no effect on speed of movement of mitochondria or APP cargo, but that the fraction of motile mitochondria was significantly increased by 36% in neurons derived from CK-B knockout mice. The percentage of motile APP vesicles was not altered. CONCLUSION: CK-B activity does not directly couple to motor protein activity but cells without the enzyme increase the number of motile mitochondria, possibly as an adaptational strategy aimed to enhance mitochondrial distribution versatility in order to compensate for loss of efficiency in the cellular network for ATP distribution.

Creatine kinase-mediated ATP supply fuels actin-based events in phagocytosis.

Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous CK-B in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and beta-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor-mediated, but not Fc-gammaR-mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.

Structural and behavioural consequences of double deficiency for creatine kinases BCK and UbCKmit.

The cytosolic brain-type creatine kinase (BCK) isoform and the mitochondrial ubiquitous creatine kinase (UbCKmit) isoform are both important for the maintenance and distribution of cellular energy in neurons and astrocytes. Previously, we reported that mice deficient for BCK or UbCKmit each showed a surprisingly mild phenotype, probably due to reciprocal functional compensation by the remaining creatine kinase. This study shows that adult male mice lacking both creatine kinase isoforms (CK--/-- double knockout mice) have a reduced body weight, and demonstrate a severely impaired spatial learning in both a dry and a wet maze, lower nestbuilding activity and diminished acoustic startle reflex responses when compared to age-matched male wildtype mice with the same genetic background. In contrast, their visual and motor functions, exploration behaviour, prepulse inhibition and anxiety-related responses were not changed, suggesting no global deficit in sensorimotor function, hearing or motivation. Morphological analysis of CK--/-- double knockout brains revealed a reduction of approximately 7% in wet brain weight and hippocampal size, a approximately 15% smaller regio-inferior and relatively larger supra-pyramidal, and intra-infra-pyramidal mossy fiber areas. These results suggest that lack of both brain specific creatine kinase isoforms renders the synaptic circuitry in adult brain less efficient in coping with sensory or cognitive activity related challenges.

Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity.

Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the regulation of microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. To investigate the biological function of PTP-BL enzyme activity, we have generated mice that lack the PTP-BL PTP moiety. These PTP-BL(DeltaP/DeltaP) mice are viable and fertile and do not present overt morphological alterations. Although PTP-BL is expressed in most hematopoietic cell lineages, no alterations of thymocyte development in PTP-BL(DeltaP/DeltaP) mice could be detected. Sciatic nerve lesioning revealed that sensory nerve recovery is unaltered in these mice. In contrast, a very mild but significant impairment of motor nerve repair was observed. Our findings exclude an essential role for PTP-BL as a phosphotyrosine phosphatase and rather are in line with a role as scaffolding or anchoring molecule.

Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions.

Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand management of proliferating cells, AK1 and AK1beta were expressed in mouse neuroblastoma N2a cells and in human colon carcinoma SW480 cells. Glucose deprivation, galactose feeding, and metabolic inhibitor tests revealed a differential energy dependency for these two cell lines. N2a cells showed a faster proliferation rate and strongest coupling to mitochondrial activity, SW480 proliferation was more dependent on glycolysis. Despite these differences, ectopic expression of AK1 or AK1beta did not affect their growth characteristics under normal conditions. Also, no differential effects were seen under metabolic stress upon treatment with mitochondrial and glycolytic inhibitors in in vitro culture or in solid tumors grown in vivo. Although many intimate connections have been revealed between cell death and metabolism, our results suggest that AK1- or AK1beta-mediated high-energy phosphoryl transfer is not a modulating factor in the survival of tumor cells during episodes of metabolic crisis.

Mice lacking the UbCKmit isoform of creatine kinase reveal slower spatial learning acquisition, diminished exploration and habituation, and reduced acoustic startle reflex responses.

Brain-type creatine kinases B-CK (cytosolic) and UbCKmit (mitochondrial) are considered important for the maintenance and distribution of cellular energy in the central nervous system. Previously, we have demonstrated an abnormal behavioral phenotype in mice lacking the B-CK creatine kinase isoform, regarding exploration, habituation, seizure susceptibility and spatial learning. The phenotype in these mice was associated with histological adaptations in the hippocampal mossy fiber field size. Here, mice lacking the ubiquitous mitochondrial creatine kinase isoform (UbCKmit-/- mice) showed, when subjected to a similar battery of behavioral tasks, diminished open field habituation and slower spatial learning acquisition in the Morris water maze task, but normal sensory or motor functions. A reduced acoustic startle response, higher threshold, and lack of prepulse inhibition were observed in UbCKmit-/- mice, suggesting that the unconditioned reflexive responsiveness is not optimal. Our findings suggest a role for mitochondrial CK-mediated high-energy phosphoryl transfer in synaptic signalling in the acoustic signal response network and hippocampal-dependent learning circuitry of brain. Finally, we demonstrate that UbCKmit has a widespread occurrence in the cell soma of neuronal nuclei along the rostro-caudal axis of the brain, i.e. cortex, midbrain, hindbrain, cerebellum and brainstem, similar to the occurrence of B-CK. This may explain the similarity of phenotypes in mice lacking B-CK or UbCKmit. We predict that the remaining functional intactness of the cytosolic B-CK reaction and perhaps the compensatory role of other phosphoryl transfer systems are sufficient to sustain the energy requirements for basic sensory, motor and physiological activities in UbCKmit-/- mice.

MR spectroscopy of muscle and brain in guanidinoacetate methyltransferase (GAMT)-deficient mice: validation of an animal model to study creatine deficiency.

As a model for guanidinoacetate methyltransferase (GAMT) deficiency in humans, a gene knockout mouse model was generated. Here we report on several metabolic abnormalities in these mice, observed by in vivo and in vitro MR spectroscopy. In (1)H MR spectra of brain and hindleg muscle a clearly reduced signal of creatine (Cr) was observed in GAMT-deficient (GAMT-/-) animals. Analysis of the (1)H MR spectra of GAMT-/- brain indicated little or no increase of a signal for guanidinoacetate (Gua). In proton MR spectra of muscle, a broad signal of low intensity was observed for Gua. However, substantial Gua accumulation in intact muscle tissue was unequivocally confirmed in high-resolution magic angle spinning spectra, in which the Gua signal was resolved as one clear sharp singlet. In (31)P MR analysis of brain and hindleg muscle a strongly reduced phosphocreatine (PCr) content was shown. In addition, a signal of phosphorylated Gua at 0.5 ppm upfield of PCr was observed, with much higher intensity in muscle than in brain. This signal decreased when ischemia was applied to the muscle and recovered after ischemia was released. Overall, the in vivo (31)P and (1)H MR spectroscopy of GAMT-/- mice is similar to that of human GAMT deficiency. This opens up new avenues for the fundamental study of tissue-type dependence of creatine synthesis and transport and for diagnostic and therapeutic aspects of creatine deficiencies in humans.

Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility.

Creatine kinases are important in maintaining cellular-energy homeostasis, and neuroprotective effects have been attributed to the administration of creatine and creatine-like compounds. Herein we examine whether ablation of the cytosolic brain-type creatine kinase (B-CK) in mice has detrimental effects on brain development, physiological integrity or task performance. Mice deficient in B-CK (B-CK-/-) showed no gross abnormalities in brain anatomy or mitochondrial ultrastructure, but had a larger intra- and infrapyramidal mossy fibre area. Nuclear magnetic resonance spectroscopy revealed that adenosine triphosphate (ATP) and phosphocreatine (PCr) levels were unaffected, but demonstrated an apparent reduction of the PCr left arrow over right arrow ATP phosphorus exchange capacity in these mice. When assessing behavioural characteristics B-CK-/- animals showed diminished open-field habituation. In the water maze, adult B-CK-/- mice were slower to learn, but acquired the spatial task. This task performance deficit persisted in 24-month-old, aged B-CK-/- mice, on top of the age-related memory decline normally seen in old animals. Finally, a delayed development of pentylenetetrazole-induced seizures (creating a high-energy demand) was observed in B-CK-/- mice. It is suggested that the persistent expression of the mitochondrial isoform ubiquitous mitochondrial CK (UbCKmit) in the creatine/phospho-creatine shuttle provides compensation for the loss of B-CK in the brain. Our studies indicate a role for the creatine-phosphocreatine/CK circuit in the formation or maintenance of hippocampal mossy fibre connections, and processes that involve habituation, spatial learning and seizure susceptibility. However, for fuelling of basic physiological activities the role of B-CK can be compensated for by other systems in the versatile and robust metabolic-energy network of the brain.