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.

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.