The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT-/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT-/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT-/y mice. Similarly, CrT-/y neurons and P10 CrT-/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.

The effect of a short-term creatine supplementation on hindlimb suspension (HS)-induced muscle atrophy was investigated. Creatine monohydrate (5 g/kg b.w. per day) or placebo, divided in 2 daily doses, was given by oral gavage for 5 days. Rats were maintained in HS with dietary supplementation concomitantly for 5 days. Body weight, soleus and EDL muscle masses, and cross-sectional areas (CSA) of the muscle fibers were measured. Signaling pathways associated with skeletal muscle mass regulation (FST, MSTN, FAK, IGF-1, MGF, Akt, mTOR, atrogin-1, and MuRF1 expressions, and Akt, S6, GSK3B, and 4EBP1 proteins) were evaluated in the muscles. Soleus muscle exhibited more atrophy than the EDL muscle due to HS. Creatine supplementation attenuated the decrease of wet weight and increased p-4EBP1 protein in the EDL muscle of HS rats. Also, creatine increased mTOR and atrogin-1 expressions in the same muscle and condition. In the absence of HS, creatine supplementation increased FAK and decreased MGF expressions in the EDL muscle. Creatine attenuated the increase in FST expression due to HS in the soleus muscle. MuRF1 expression increased in the soleus muscle due to creatine supplementation in HS animals whereas atrogin-1 expression increased still further in this group compared with untreated HS rats. In conclusion, short-term creatine supplementation changed protein metabolism signaling in soleus and EDL muscles. However, creatine supplementation only slightly attenuated the mass loss of both muscles and did not prevent the CSA reduction and muscle strength decrease induced by HS for 5 days.

Attenuation of age-related sarcopenia by creatine supplementation has been equivocal. In this study, plantaris muscles of young (Y; 5m) and aging (A; 24m) Fisher 344 rats underwent four weeks of either control (C), creatine supplementation (Cr), surgical overload (O), or overload plus creatine (OCr). Creatine alone had no effect on muscle fiber cross-sectional area (CSA) or heat shock protein (HSP70) and increased myonuclear domain (MND) only in young rats. Overload increased CSA and HSP70 content in I and IIA fibers, regardless of age, and MND in IIA fibers of YO rats. CSA and MND increased in all fast fibers of YOCr, and CSA increased in I and IIA fibers of AOCr. OCR did not alter HSP70, regardless of age. MND did not change in aging rats, regardless of treatment. These data indicate creatine alone had no significant effect. Creatine with overload produced no additional hypertrophy relative to overload alone and attenuated overload-induced HSP70 expression.

Creatine is important for energy metabolism, yet excitable cells such as cardiomyocytes do not synthesize creatine and rely on uptake via a specific membrane creatine transporter (CrT; SLC6A8). This process is tightly controlled with downregulation of CrT upon continued exposure to high creatine via mechanisms that are poorly understood. Our aim was to identify candidate endogenous CrT inhibitors. In 3T3 cells overexpressing the CrT, creatine uptake plateaued at 3 h in response to 5 mM creatine but peaked 33% higher (P < 0.01) in the presence of cycloheximide, suggesting CrT regulation depends on new protein synthesis. Global gene expression analysis identified thioredoxin-interacting protein (Txnip) as the only significantly upregulated gene (by 46%) under these conditions (P = 0.036), subsequently verified independently at mRNA and protein levels. There was no change in Txnip expression with exposure to 5 mM taurine, confirming a specific response to creatine rather than osmotic stress. Small-interfering RNA against Txnip prevented Txnip upregulation in response to high creatine, maintained normal levels of creatine uptake, and prevented downregulation of CrT mRNA. These findings were relevant to the in vivo heart since creatine-deficient mice showed 39.71% lower levels of Txnip mRNA, whereas mice overexpressing the CrT had 57.6% higher Txnip mRNA levels and 28.7% higher protein expression compared with wild types (mean myocardial creatine concentration 124 and 74 nmol/mg protein, respectively). In conclusion, we have identified Txnip as a novel negative regulator of creatine levels in vitro and in vivo, responsible for mediating substrate feedback inhibition and a potential target for modulating creatine homeostasis.

It is well established that the immature myocardium preferentially utilises non-oxidative energy-generating pathways. It exhibits low energy-transfer capacity via the creatine kinase (CK) shuttle, reflected in phosphocreatine (PCr), total creatine and CK levels that are much lower than those of adult myocardium. The mechanisms leading to gradually increasing energy transfer capacity during maturation are poorly understood. Creatine is not synthesised in the heart, but taken up exclusively by the action of the creatine transporter protein (CrT). To determine whether this transporter is ontogenically regulated, the present study serially examined CrT gene expression pattern, together with creatine uptake kinetics and resulting myocardial creatine levels, in rats over the first 80 days of age.

Due to the pivotal role of microfilament in cancer cells, targeting microfilaments with cytochalasins is considered a promising anticancer strategy. Here, we obtained cytochalasin Q (CQ) from Xylaria sp. DO1801, the endophytic fungi from the root of plant Damnacanthus officinarum, and discovered its anti-melanoma activity in vivo and in vitro attributing to microfilament depolymerization. Mechanistically, CQ directly bound to and inactivated creatine kinase B (CKB), an enzyme phosphorylating creatine to phosphocreatine (PCr) and regenerating ATP to cope with high energy demand, and then inhibited the creatine metabolism as well as cytosolic glycolysis in melanoma cells. Preloading PCr recovered ATP generation, reversed microfilament depolymerization and blunted anti-melanoma efficacy of CQ. Knockdown of CKB resulted in reduced ATP level, perturbed microfilament, inhibited proliferation and induced apoptosis, and manifested lower sensitivity to CQ. Further, we found that either CQ or CKB depletion suppressed the PI3K/AKT/FoxO1 pathway, whereas 740Y-P, a PI3K agonist, elevated protein expression of CKB suppressed by CQ. Taken together, our study highlights the significant anti-melanoma effect and proposes a PI3K/AKT/FoxO1/ CKB feedback circuit for the activity of CQ, opening new opportunities for current chemotherapy.

Creatine deficiency syndrome (CDS) comprises three separate enzyme deficiencies with overlapping clinical presentations: arginine:glycine amidinotransferase (GATM gene, glycine amidinotransferase), guanidinoacetate methyltransferase (GAMT gene), and creatine transporter deficiency (SLC6A8 gene, solute carrier family 6 member 8). CDS presents with developmental delays/regression, intellectual disability, speech and language impairment, autistic behaviour, epileptic seizures, treatment-refractory epilepsy, and extrapyramidal movement disorders; symptoms that are also evident in children with autism. The objective of the study was to test the hypothesis that genetic variability in creatine metabolism genes is associated with autism. We sequenced GATM, GAMT and SLC6A8 genes in 166 patients with autism (coding sequence, introns and adjacent untranslated regions). A total of 29, 16 and 25 variants were identified in each gene, respectively. Four variants were novel in GATM, and 5 in SLC6A8 (not present in the 1000 Genomes, Exome Sequencing Project (ESP) or Exome Aggregation Consortium (ExAC) databases). A single variant in each gene was identified as non-synonymous, and computationally predicted to be potentially damaging. Nine variants in GATM were shown to have a lower minor allele frequency (MAF) in the autism population than in the 1000 Genomes database, specifically in the East Asian population (Fisher's exact test). Two variants also had lower MAFs in the European population. In summary, there were no apparent associations of variants in GAMT and SLC6A8 genes with autism. The data implying there could be a lower association of some specific GATM gene variants with autism is an observation that would need to be corroborated in a larger group of autism patients, and with sub-populations of Asian ethnicities. Overall, our findings suggest that the genetic variability of creatine synthesis/transport is unlikely to play a part in the pathogenesis of autism spectrum disorder (ASD) in children.

Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H(2)O(2). In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

Creatine plays an important role in the cell as an energy buffer. As the energy system is a basic element of the organism it may possibly contribute to differences between rainbow trout strains selected for the traits growth and robustness, respectively. The cDNA sequences of creatine-related genes encoding glycine amidinotransferase (GATM), guanidinoacetate N-methyltransferase (GAMT), creatine kinase muscle-type (CKM) and creatine transporter 1 (CT1, encoded by gene solute carrier family 6, member 8 (SLC6A8)) were characterized in rainbow trout. Transcripts of the respective genes were quantified in kidney, liver, brain and skeletal muscle in both trout strains that had been acclimated to different temperatures. Several differences between the compared trout strains were found as well as between temperatures indicating that the energy system may contribute to differences between both strains. In addition to that, the expression data showed clear differences between the creatine system in rainbow trout and mammals, as the spatial distribution of the enzyme-encoding gene expression was clearly different from the patterns described for mammals. In rainbow trout, creatine synthesis seems to take place to a big extent in the skeletal muscle.

Ischemia-reperfusion injury (IRI) is one of the factors limiting the success of lung transplantation (LTx). IRI increases death risk after transplantation through innate immune system activation and inflammation induction. Some studies have shown that creatine (Cr) protects tissues from ischemic damage by its antioxidant action. We evaluated the effects of Cr supplementation on IRI after unilateral LTx in rats. Sixty-four rats were divided into four groups: water + 90 min of ischemia; Cr + 90 min of ischemia; water + 180 min of ischemia; and Cr + 180 min of ischemia. Donor animals received oral Cr supplementation (0.5 g/kg/day) or vehicle (water) for five days prior to LTx. The left lung was exposed to cold ischemia for 90 or 180 min, followed by reperfusion for 2 h. We evaluated the ventilatory mechanics and inflammatory responses of the graft. Cr-treated animals showed a significant decrease in exhaled nitric oxide levels and inflammatory cells in blood, bronchoalveolar lavage fluid and lung tissue. Moreover, edema, cell proliferation and apoptosis in lung parenchyma were reduced in Cr groups. Finally, TLR-4, IL-6 and CINC-1 levels were lower in Cr-treated animals. We concluded that Cr caused a significant decrease in the majority of inflammation parameters evaluated and had a protective effect on the IRI after LTx in rats.

Organisms obtain creatine from their diet or by de novo synthesis via AGAT (L-arginine:glycine amidinotransferase) and GAMT (Guanidinoacetate N-methyltrasferase) in kidney and liver, respectively. AGAT also synthesizes homoarginine (hArg), low levels of which predict poor outcomes in human cardiovascular disease, while supplementation maintains contractility in murine heart failure. However, the expression pattern of AGAT has not been systematically studied in mouse tissues and nothing is known about potential feedback interactions between creatine and hArg. Herein, we show that C57BL/6J mice express AGAT and GAMT in kidney and liver respectively, whereas pancreas was the only organ to express appreciable levels of both enzymes, but no detectable transmembrane creatine transporter (Slc6A8). In contrast, kidney, left ventricle (LV), skeletal muscle and brown adipose tissue must rely on creatine transporter for uptake, since biosynthetic enzymes are not expressed. The effects of creatine and hArg supplementation were then tested in wild-type and AGAT knockout mice. Homoarginine did not alter creatine accumulation in plasma, LV or kidney, whereas in pancreas from AGAT KO, the addition of hArg resulted in higher levels of tissue creatine than creatine-supplementation alone (P < 0.05). AGAT protein expression in kidney was downregulated by creatine supplementation (P < 0.05), consistent with previous reports of end-product repression. For the first time, we show that hArg supplementation causes a similar down-regulation of AGAT protein (P < 0.05). These effects on AGAT were absent in the pancreas, suggesting organ specific mechanisms of regulation. These findings highlight the potential for interactions between creatine and hArg that may have implications for the use of dietary supplements and other therapeutic interventions.

Creatine is an ergogenic compound used by athletes to enhance performance. Supplementation with creatine monohydrate (CM) has been suggested for musculoskeletal and neurological disorders. Until now, little is known about its pharmacokinetic profile. Our objective was to determine the oral bioavailability of CM and the influence of dose on oral absorption. Rats were dosed orally with low dose (10 mg/kg) or high dose (70 mg/kg) 13C-labeled CM. Blood samples were removed at various time points. Muscle and brain tissue were collected at the conclusion of the study. Plasma and tissue levels of 13C-labeled creatine were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Physiologically based pharmacokinetic (PBPK) models of CM were built using GastroPlus™. These models were used to predict the plasma concentration-time profiles of creatine hydrochloride (CHCL), which has improved aqueous solubility compared to CM. Absolute oral bioavailability for low dose CM was 53% while high dose CM was only 16%. The simulated Cmax of 70 mg/kg CHCL was around 35 μg/mL compared to 14 μg/mL for CM with a predicted oral bioavailability of 66% with CHCL compared to 17% with CM. Our results suggest that the oral bioavailability of CM is less than complete and subject to dose and that further examination of improved dosage formulations of creatine is warranted.

Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine's positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks. Nuclear and mitochondrial DNAs from oxidatively-injured human umbilical vein endothelial cells were analyzed. Creatine supplementation showed significant genoprotective activity on mitochondrial DNA. This evidence suggests that creatine may play an important role in mitochondrial genome stability in that it could normalize mitochondrial mutagenesis and its functional consequences. Thus, creatine supplementation could be used to prevent or ameliorate diseases related to mitochondrial DNA mutations, and possibly to delay aging.

A great deal of evidence has been gathered on the use of creatine as an ergogenic supplement. Recent studies show greater benefits when creatine ingestion is performed close in time to training, but few studies tackle the way that circadian rhythms could influence creatine consumption. The aim of this study was therefore to observe the influence circadian rhythms exert on sports performance after creatine supplementation. Our method involved randomly assigning fourteen women players of a handball team into two groups in a single-blind study: one that consumed the supplement in the morning and one that consumed it in the evening, with both groups following a specific training program. After twelve weeks, the participants exhibited a decreased fat percentage, increased body weight and body water, and improved performance, with these results being very similar in the two groups. It is therefore concluded that, although circadian rhythms may influence performance, these appear not to affect creatine supplementation, as creatine is stored intramuscularly and is available for those moments of high energy demand, regardless of the time of day.

Consuming more creatine may be associated with an increased risk of renal dysfunction, yet this link remains poorly addressed at the population level. Using 2017-2018 NHANES data, the current study found that the odds ratio for having failing kidneys in 2,955 U.S adults consuming ≥2.0 g/day of dietary creatine compared to low-intake counterparts (<1.0 g/day) was 0.74 (95% CI from 0.39 to 1.38), indicating no significant association between dietary creatine intake and kidney dysfunction.

Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure.

Creatine is an organic compound which is utilized in biological activities, especially for adenosine triphosphate (ATP) production in the phosphocreatine system. This is a well-known biochemical reaction that is generally recognized as being mainly driven in specific parts of the body, such as the skeletal muscle and brain. However, our report shows a novel aspect of creatine utilization and ATP synthesis in innate immune cells. Creatine supplementation enhanced immune responses in neutrophils, such as cytokine production, reactive oxygen species (ROS) production, phagocytosis, and NETosis, which were characterized as antibacterial activities. This creatine-induced functional upregulation of neutrophils provided a protective effect in a murine bacterial sepsis model. The mortality rate in mice challenged with Escherichia coli K-12 was decreased by creatine supplementation compared with the control treatment. Corresponding to this decrease in mortality, we found that creatine supplementation decreased blood pro-inflammatory cytokine levels and bacterial colonization in organs. Creatine supplementation significantly increased the cellular ATP level in neutrophils compared with the control treatment. This ATP increase was due to the phosphocreatine system in the creatine-treated neutrophils. In addition, extracellular creatine was used in this ATP synthesis, as inhibition of creatine uptake abolished the increase in ATP in the creatine-treated neutrophils. Thus, creatine is an effective nutrient for modifying the immunological function of neutrophils, which contributes to enhancement of antibacterial immunity.

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.

McArdle disease (MD) is a metabolic myopathy due to myophosphorylase deficiency. We examined monocarboxylate transporters (MCT) and creatine kinase (CK) protein content in skeletal muscle from MD patients and age-matched controls to evaluate potential cellular adaptations that compensate for the loss of glycogenolysis. Our findings of higher MCT1 and mitochondrial CK suggest that proteins related to extra-muscular fuel uptake and intra-muscular energy transduction are up-regulated without change in mitochondrial mass in MD patients.

Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.