The creatine kinase system and pleiotropic effects of creatine

Abstract

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure-function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.

Fig. 1

The CK/PCr system for temporal and spatial energy buffering in cells of high and fluctuating energy requirements. Cr enters the target cells via Cr transporter (CRT). Inside the cell, PCr/Cr and ATP/ADP equilibria are adjusted by a soluble fraction of cytosolic CK isoforms (CK-c, see (3)). Another fraction of cytosolic CK (CK-g, see (2)) is specifically coupled to glycolytic enzymes (G), accepting glycolytic ATP, while mitochondrial CK isoforms (mtCK, see (1)) is coupled to adenine nucleotide translocator (ANT), thus accepting ATP exported from the matrix and generated by oxidative phosphorylation (OP). The contribution of both of these so-called microcompartments to total PCr generation depends on the cell type. The PCr thus generated is fed into the large PCr pool (up to 30 mM) that is available as a temporal or spatial energy buffer. Another fraction of cytosolic CK (CK-a, see (4)) specifically associated with subcellular sites of ATP utilization (ATPase, e.g. ATP-dependent or ATP-gated processes, ion-pumps etc.) also forms tightly coupled microcompartments regenerating the ATP utilized by the ATPase reaction in situ on the expense of PCr. The proposed CK/PCr energy shuttle or circuit connects, via highly diffusible PCr and Cr, subcellular sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). This model is based on functionally coupled, subcellular CK microcompartments, where ATP production and ATP consumption are tightly connected to CK/PCr action (Wallimann ; Wallimann and Eppenberger ; Schlattner et al. ; Wallimann et al. 2007)

Fig. 2

Spatial energy buffering by the CK/PCr: the PCr/Cr-shuttle in spermatozoa. Diffusion fluxes in sea urchin sperm were calculated from in vivo ³¹P-NMR saturation transfer NMR experiments (Kaldis et al. 1997). The diffusion flux of ADP from the sperm tail end towards the mitochondrion located at the mid piece is more than 2,000 times slower compared with that of Cr, whereas the diffusion flux of ATP from the region of the mitochondrion towards the sperm tail is roughly seven times slower than that of PCr. Thus, the PCr/Cr-shuttle is a physiological adaptation to overcome the diffusional limitations of adenosine nucleotides, especially of ADP, to facilitate long-distance energy transport, as well as high-throughput fluxes of cellular energy. A similar system has been proposed and proved to work also in the sensory hair cells of the inner ear (Shin et al. 2007) and in the polar photoreceptor cells of the retina (Wallimann et al. ; Hemmer et al. ; Linton et al. 2010) and in the sensory hair cells of the inner ear (Shin et al. 2007). (Figure adapted from Kaldis et al. 1997)

Fig. 3

Mitochondrial mtCK functions for high-energy metabolite channelling in mitochondria. In cells with oxidative metabolism, respiration (green arrow), ATP synthesis and ATP export through the inner mitochondrial membrane via adenine nucleotide transporter (ANT) are tightly coupled to trans-phosphorylation of ATP to PCr by mtCK and export of PCr into the cytosol by the outer membrane voltage-dependent anion channel (VDAC) as indicated by black arrows. In turn, Cr stimulates respiration by favoring constant supply of ADP to the matrix (black arrows), which also lowers ROS/RNS production in the intra-mitochondrial space (red arrows) and inhibits mitochondrial permeability transition. The tight coupling of substrate and product fluxes (black arrows) allows a so-called channeling of “high-energy” metabolites, with PCr being the one released into the cytosol, and ATP/ADP being mainly recycled within the mitochondria. The structural basis of these mtCK microcompartments are proteolipid complexes containing either VDAC, octameric mtCK and ANT in the peripheral intermembrane space (as shown) or octameric mtCK and ANT in the cristae (not shown). These proteolipid complexes are maintained by mtCK interactions with anionic phospholipids and VDAC in the outer membrane, and with cardiolipin and thus indirectly with cardiolipin-associated ANT in the inner membrane (see cardiolipoin patches). In cases of a less coupled mtCK microcompartment, e.g. after impairment of mtCK function by oxidative damage, there is partial direct ATP/ADP exchange with the cytosol (blue arrows). (Figure adapted from Kaldis et al. ; Meyer et al. ; Schlattner et al. ; Schlattner et al. 2011) (The different fluxes are indicated by coloured arrows in the figure)

Fig. 4

Mitochondrial permeability transition is inhibited by CK substrates. At a concentration of 10 mM, the CK substrates creatine (Cr) and cyclocreatine (cCr), Cr-analogon, inhibit MTP in isolated mouse liver mitochondria to a comparable degree as 1 μM cyclosporin A (CSA), the gold standard for MPT inhibition (Dolder et al. 2003). The Cr-analogon guanidinopropionic acid (GPA) that is not accepted as a substrate by the CK reaction has no effect as compared to control without additions (none). Isolated liver mitochondria from transgenic mice expressing uMtCK in their liver were analysed by a swelling (light scattering) assay. They were energized with glutamate/malate in presence of 2mM Mg²⁺ and then challenged by 120 μM Ca²⁺ where indicated. (Figure taken from Dolder et al. with permission)