Creatine Uptake Research Articles

Abstract 5863: RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression

Abstract Background: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide with more than 140,000 patients diagnosed and nearly 50,000 deaths annually in the U.S. alone. Roughly 60% of patients present with locally advanced or distant metastatic disease, with the liver being a primary site of metastatic colonization. Creatine metabolism has been implicated in colon cancer progression and metastatic colonization of the liver. Metastatic colon cancer cells upregulate and release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high-energy metabolite phosphocreatine. Phosphocreatine is imported via the creatine transporter SLC6a8. Intracellular phosphocreatine can be converted to ATP to fuel the survival of metastatic cancer cells within the hypoxic hepatic microenvironment. Consistent with this finding, genetic depletion of SLC6a8 in colon and pancreatic cancer cell lines significantly reduced liver colonization in mouse xenograft models. Results: We herein demonstrate that the novel small molecule RGX-202 is a robust inhibitor of creatine uptake in cancer cells, both in vitro and in vivo. Oral administration of RGX-202 induced apoptosis of colon cancer cells in vivo, and significantly suppressed colon cancer liver metastatic colonization and primary tumor growth, both in KRAS wild-type and KRAS mutant colon cancer cell lines as well as in human PDX mouse models. Using genetic studies, these effects were found to be dependent on tumoral expression of SLC6a8. In addition, combination treatment of the CT26 syngeneic colon cancer mouse model with 5-FU resulted in synergistic antitumor activity, with complete tumor regressions observed in more than 40% of treated mice. Similarly, combination treatment of the KPC syngeneic mouse model with gemcitabine significantly reduced the growth of primary pancreatic tumors. Definitive 28-day GLP toxicology and pharmacokinetics studies of RGX-202 are currently ongoing. Preliminary observations suggest good tolerability in several animal species with a favorable pharmacokinetic profile, including bioavailability. Conclusion: These results strongly support clinical development of RGX-202 in patients with gastrointestinal cancers, including colorectal and pancreatic cancer, both as monotherapy and in combination with standard-of-care treatment. Citation Format: Isabel Kurth, Celia Andreu, Shugaku Takeda, Helen Tian, Foster Gonsalves, Katya Leites, Subhasree Sridhar, Jia Min Loo, Rob Busby, Sohail Tavazoie, Masoud Tavazoie. RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5863.

Mechanism of obesity suppression by adipose tissue creatine energetics

The increase in obesity worldwide has led to a surge in the prevalence of type 2 diabetes, cardiovascular disease and many cancers. The capacity of thermogenic (brown and beige) fat to dissipate chemical energy as heat offers tremendous potential to combat obesity. Exploiting thermogenesis to regulate body weight requires a precise molecular understanding of its control at the level of mitochondria.We have previously demonstrated that creatine drives energy expenditure in adipose tissue by stimulating a futile cycle of mitochondrial ATP turnover. Our recent findings show that genetic deletion of the rate‐limiting enzyme of creatine synthesis (Gatm) results in rapid‐onset obesity due to the suppression of elevated energy expenditure that occurs in response to high calorie feeding. This is paralleled by a blunted capacity for β3‐adrenergic activation of metabolic rate. Moreover, abolishing adipocyte creatine uptake, by deleting the creatine transporter (CrT), similarly impairs adrenergic thermogenesis and causes obesity. These results provide strong in vivo genetic support for a role of adipose tissue creatine metabolism in the activation of energy expenditure and the defense against diet‐induced obesity. However, the mechanism underlying creatine‐dependent thermogenesis is not fully understood.We have now identified the primary Creatine Kinase (CK) isoform that controls thermogenic respiration in brown fat. Using quantitative proteomics from purified brown adipocytes and brown adipocyte mitochondria, we find brain‐type CK (B‐CK) to be the most abundant CK in murine brown fat. Surprisingly a subset of B‐CK is targeted to mitochondria. Mitochondria from primary brown adipocytes have robust capacity for creatine‐dependent thermogenic respiration, and knockdown of B‐CK impairs this process. Notably, B‐CK is the most abundance CK isoform in purified human subcutaneous adipocytes and its expression is significantly lower in insulin‐resistant compared to insulin‐sensitive patients. Therefore, B‐CK is the first molecular handle that should result in the identification of additional components regulating creatine‐dependent thermogenesis. In addition, adipose tissue creatine energetics is correlated with healthy glucose homeostasis in humans.Support or Funding InformationCanadian Institutes of Health Research postdoctoral fellowship and NIDDK, NIH Pathway to Independence (K99/R00), K99DK114528 (to L.K.)NIH grant (DK31405) and the JPB Foundation (to B.M.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional assessment of creatine transporter in control and X-linked SLC6A8-deficient fibroblasts

Creatine transporter is currently the focus of renewed interest with emerging roles in brain neurotransmission and physiology, and the bioenergetics of cancer metastases. We here report on amendments of a standard creatine uptake assay which might help clinical chemistry laboratories to extend their current range of measurements of creatine and metabolites in body fluids to functional enzyme explorations. In this respect, short incubation times and the use of a stable-isotope-labeled substrate (D3-creatine) preceded by a creatine wash-out step from cultured fibroblast cells by removal of fetal bovine serum (rich in creatine) from the incubation medium are recommended. Together, these measures decreased, by a first order of magnitude, creatine concentrations in the incubation medium at the start of creatine-uptake studies and allowed to functionally discriminate between 4 hemizygous male and 4 heterozygous female patients with X-linked SLC6A8 deficiency, and between this cohort of eight patients and controls. The functional assay corroborated genetic diagnosis of SLC6A8 deficiency. Gene anomalies in our small cohort included splicing site (c.912G > A [p.Ile260_Gln304del], c.778-2A > G and c.1495 + 2 T > G), substitution (c.407C > T) [p.Ala136Val] and deletion (c.635_636delAG [p.Glu212Valfs*84] and c.1324delC [p.Gln442Lysfs*21]) variants with reduced creatine transporter function validating their pathogenicity, including that of a previously unreported c.1324delC variant. The present assay adaptations provide an easy, reliable and discriminative manner for exploring creatine transporter activity and disease variations. It might apply to drug testing or other evaluations in the genetic and metabolic horizons covered by the emerging functions of creatine and its transporter, in a way, however, requiring and completed by additional studies on female patients and blood-brain barrier permeability properties of selected compounds. As a whole, the proposed assay of creatine transporter positively adds to currently existing measurements of this transporter activity, and determining on a large scale the extent of its exact suitability to detect female patients should condition in the future its transfer in clinical practice.

Creatine-electrolyte supplementation improves repeated sprint cycling performance: A double blind randomized control study

BackgroundCreatine supplementation is recommended as an ergogenic aid to improve repeated sprint cycling performance. Furthermore, creatine uptake is increased in the presence of electrolytes. Prior research examining the effect of a creatine-electrolyte (CE) supplement on repeated sprint cycling performance, however, did not show post-supplementation improvement. The purpose of this double blind randomized control study was to investigate the effect of a six-week CE supplementation intervention on overall and repeated peak and mean power output during repeated cycling sprints with recovery periods of 2 min between sprints.MethodsPeak and mean power generated by 23 male recreational cyclists (CE group: n = 12; 24.0 ± 4.2 years; placebo (P) group: n = 11; 23.3 ± 3.1 years) were measured on a Velotron ergometer as they completed five 15-s cycling sprints, with 2 min of recovery between sprints, pre- and post-supplementation. Mixed-model ANOVAs were used for statistical analyses.ResultsA supplement-time interaction showed a 4% increase in overall peak power (pre: 734 ± 75 W; post: 765 ± 71 W; p = 0.040; ηp2 = 0.187) and a 5% increase in overall mean power (pre: 586 ± 72 W; post: 615 ± 74 W; p = 0.019; ηp2 = 0.234) from pre- to post-supplementation for the CE group. For the P group, no differences were observed in overall peak (pre: 768 ± 95 W; post: 772 ± 108 W; p = 0.735) and overall mean power (pre: 638 ± 77 W; post: 643 ± 92 W; p = 0.435) from pre- to post-testing. For repeated sprint analysis, peak (pre: 737 ± 88 W; post: 767 ± 92 W; p = 0.002; ηp2 = 0.380) and mean (pre: 650 ± 92 W; post: 694 ± 87 W; p < 0.001; ηp2 = 0.578) power output were significantly increased only in the first sprint effort in CE group from pre- to post-supplementation testing. For the P group, no differences were observed for repeated sprint performance.ConclusionA CE supplement improves overall and repeated short duration sprint cycling performance when sprints are interspersed with adequate recovery periods.

Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation.

Creatine (Cr), phosphocreatine (PCr), and creatine kinases (CK) comprise an energy shuttle linking ATP production in mitochondria with cellular consumption sites. Myocytes cannot synthesize Cr: these cells depend on uptake across the cell membrane by a specialized creatine transporter (CrT) to maintain intracellular Cr levels. Hypoxia interferes with energy metabolism, including the activity of the creatine energy shuttle, and therefore affects intracellular ATP and PCr levels. Here, we report that exposing cultured cardiomyocytes to low oxygen levels rapidly diminishes Cr transport by decreasing V max and K m. Pharmacological activation of AMP‐activated kinase (AMPK) abrogated the reduction in Cr transport caused by hypoxia. Cr supplementation increases ATP and PCr content in cardiomyocytes subjected to hypoxia, while also significantly augmenting the cellular adaptive response to hypoxia mediated by HIF‐1 activation. Our results indicate that: (1) hypoxia reduces Cr transport in cardiomyocytes in culture, (2) the cytoprotective effects of Cr supplementation are related to enhanced adaptive physiological responses to hypoxia mediated by HIF‐1, and (3) Cr supplementation increases the cellular ATP and PCr content in RNCMs exposed to hypoxia.

Potential for use of creatine supplementation following mild traumatic brain injury.

There is significant overlap between the neuropathology of mild traumatic brain injury (mTBI) and the cellular role of creatine, as well as evidence of neural creatine alterations after mTBI. Creatine supplementation has not been researched in mTBI, but shows some potential as a neuroprotective when administered prior to or after TBI. Consistent with creatine’s cellular role, supplementation reduced neuronal damage, protected against the effects of cellular energy crisis and improved cognitive and somatic symptoms. A variety of factors influencing the efficacy of creatine supplementation are highlighted, as well as avenues for future research into the potential of supplementation as an intervention for mTBI. In particular, the slow neural uptake of creatine may mean that greater effects are achieved by pre-emptive supplementation in at-risk groups.

Impaired muscle uptake of creatine in spinal and bulbar muscular atrophy.

ObjectiveThe aim of this study was to explore the pathomechanism underlying the reduction of serum creatinine (Cr) concentrations in spinal and bulbar muscular atrophy (SBMA).MethodsWe evaluated blood chemistries, motor function, and muscle mass measured by dual‐energy X‐ray absorptiometry in male subjects with SBMA (n = 65), amyotrophic lateral sclerosis (ALS; n = 27), and healthy controls (n = 25). We also examined the intramuscular concentrations of creatine, a precursor of Cr, as well as the protein and mRNA expression levels of the creatine transporter (SLC6A8) in autopsy specimens derived from subjects who had SBMA and ALS and disease controls. Furthermore, we measured the mRNA expression levels of SLC6A8 in cultured muscle cells (C2C12) transfected with the polyglutamine‐expanded androgen receptor (AR‐97Q).ResultsSerum Cr concentrations were significantly lower in subjects with SBMA than in those with ALS (P < 0.001), despite similar muscle mass values. Intramuscular creatine concentrations were also lower in with the autopsied specimen of SBMA subjects than in those with ALS subjects (P = 0.018). Moreover, the protein and mRNA expression levels of muscle SLC6A8 were suppressed in subjects with SBMA. The mRNA levels of SLC6A8 were also suppressed in C2C12 cells bearing AR‐97Q.InterpretationThese results suggest that low serum Cr concentration in subjects with SBMA is caused by impaired muscle uptake of creatine in addition to being caused by neurogenic atrophy. Given that creatine serves as an energy source in skeletal muscle, increasing muscle creatine uptake is a possible therapeutic approach for treating SBMA.

Fenugreek increases insulin-stimulated creatine content in L6C11 muscle myotubes.

Creatine uptake by muscle cells is increased in the presence of insulin. Accordingly, compounds with insulin-like actions may also augment creatine uptake. The aim of this study was to investigate whether Trigonella foenum-graecum (fenugreek), an insulin mimetic, increases total intracellular creatine levels in vitro. Total cellular creatine content was measured fluorometrically in L6C11 muscle myotubes treated for 1, 4, and 24h with 0.5mM creatine (CR), CR and 20μg/mL fenugreek seed extract (CR+FEN), CR and 100nM insulin (CR+INS), and CR+INS+FEN (n=6 per treatment group). Alterations in the expression of the sodium- and chloride-dependent creatine transporter, SLC6A8, and key signaling proteins in the PI3-K/Akt pathway were determined. Compared to control (CON), CR+INS+FEN increased total creatine content after 4h (P<0.05), whereas all conditions increased SLC6A8 protein expression above CON at this time (P<0.05). Changes in insulin signaling were demonstrated via increases in AktThr308 phosphorylation, with CR+INS>CON and CR at 1h (P<0.05) and with CR+INS+FEN>CON, CR, and CR+INS at 4h (P<0.05). In contrast, no changes in PKCζ/λ or GLUT4 phosphorylation were detected. Fenugreek, when combined with insulin, modulates creatine content via a mechanism which is independent of the activity of SLC6A8, suggesting that an alternative mechanism is responsible for the regulation and facilitation of insulin-mediated creatine uptake in skeletal muscle cells.

Creatine biosynthesis and transport in health and disease.

Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.

The effects of creatine monohydrate supplementation on creatine transporter activity and creatine metabolism in resistance trained males

Background Oral creatine supplementation is known to provide numerous benefits, including increases in lean muscle mass, muscular strength, and enhanced performance in various athletic capacities. The creatine transporter is a transmembrane protein that mediates the entry of creatine from the circulation into the muscle cell. Little is understood about the importance of the creatine transporter in controlling the uptake and regulation of creatine within human skeletal muscle. The purpose of this study was to conduct a preliminary examination of the effects of a typical creatine monohydrate supplementation regimen on the activity of the creatine transporter at the transcriptional and translational levels in resistance-trained males.

Effects of a traditionally-dosed creatine supplementation protocol and resistance training on the skeletal muscle uptake and whole-body metabolism and retention of creatine in males

Background A typical oral creatine supplementation regimen involving a 5-7 day “loading phase” of 20-25 grams/day followed by a “maintenance phase” of 5-7 grams/day is typically considered as necessary to adequately saturate skeletal muscle as a lesser dose of creatine is insufficient in doing so. This rationale also assumes that the majority, if not all, of the creatine ingested at this dosage is fully utilized by skeletal muscle as a phosphate reservoir in which to re-synthesize ATP during high-intensity, short-term exercise. The purpose of this study was simply to determine the effects of this “typical” creatine dosing strategy previously mentioned on skeletal muscle creatine uptake as well as the whole-body metabolism and retention of creatine in males while engaged in resistance training.

Estimated carrier frequency of creatine transporter deficiency in females in the general population using functional characterization of novel missense variants in the SLC6A8 gene

Creatine transporter deficiency (CRTR-D) is an X-linked inherited disorder of creatine transport. All males and about 50% of females have intellectual disability or cognitive dysfunction. Creatine deficiency on brain proton magnetic resonance spectroscopy and elevated urinary creatine to creatinine ratio are important biomarkers. Mutations in the SLC6A8 gene occur de novo in 30% of males. Despite reports of high prevalence of CRTR-D in males with intellectual disability, there are no true prevalence studies in the general population. To determine carrier frequency of CRTR-D in the general population we studied the variants in the SLC6A8 gene reported in the Exome Variant Server database and performed functional characterization of missense variants. We also analyzed synonymous and intronic variants for their predicted pathogenicity using in silico analysis tools.Nine missense variants were functionally analyzed using transient transfection by site-directed mutagenesis with In-Fusion HD Cloning in HeLa cells. Creatine uptake was measured by liquid chromatography tandem mass spectrometry for creatine measurement.The c.1654G>T (p.Val552Leu) variant showed low residual creatine uptake activity of 35% of wild type transfected HeLa cells and was classified as pathogenic. Three variants (c.808G>A; p.Val270Met, c.942C>G; p.Phe314Leu and c.952G>A; p.Ala318Thr) were predicted to be pathogenic based on in silico analysis, but proved to be non-pathogenic by our functional analysis. The estimated carrier frequency of CRTR-D was 0.024% in females in the general population.We recommend functional studies for all novel missense variants by transient transfection followed by creatine uptake measurement by liquid chromatography tandem mass spectrometry as fast and cost effective method for the functional analysis of missense variants in the SLC6A8 gene.

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P<0.0125) and creatine uptake (1.8 and 1.6-fold, P<0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P<0.05), resulting in a 2.2-fold (P<0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.

Post-transcriptional regulation of the creatine transporter gene: Functional relevance of alternative splicing

BackgroundAberrations in about 10–15% of X-chromosome genes account for intellectual disability (ID); with a prevalence of 1–3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter (CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8–SLC6A8C, containing intron 4 and exons 5–13, was identified. Herein, we report the identification of a novel variant — SLC6A8D, and functional relevance of these isoforms. MethodsVia (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport. ResultsSLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift). Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8, both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform resulted in a significant increase in creatine accumulation compared to CTR1 cells re-infected with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into HEK293 cells resulted in significantly higher creatine uptake. ConclusionsCTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake. General significanceProvides added insight into the mechanism(s) of creatine transport regulation.

Urinary creatine at rest and after repeated sprints in athletes: a pilot study.

Creatine plays a key role in muscle function and its evaluation is important in athletes. In this study, urinary creatine concentration was measured in order to highlight its possible significance in monitoring sprinters. The study included 51 sprinters and 25 age- and sex-matched untrained subjects as a control group. Body composition was measured and dietary intake estimated. Urine samples were collected before and after standardized physical exercise. Creatine was assessed by gas chromatography mass spectrometry. Basal urinary creatine (UC) was significantly lower in sprinters than controls (34±30 vs. 74±3 µmol/mmol creatinine, p < 0.05). UC was inversely correlated with body mass (r = −0.34, p < 0.01) and lean mass (r = −0.30, p < 0.05), and positively correlated with fat mass (r = 0.32, p < 0.05). After acute exercise, urinary creatine significantly decreased in both athletes and controls. UC is low in sprinters at rest and further decreases after exercise, most likely due to a high uptake and use of creatine by muscles, as muscle mass and physical activity are supposed to be greater in athletes than untrained subjects. Further studies are needed to test the value of urinary creatine as a non-invasive marker of physical condition and as a parameter for managing Cr supplementation in athletes.

Negative Regulation of the Creatine Transporter SLC6A8 by SPAK and OSR1

Background/Aims: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na⁺ coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. Methods: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active ^T233ESPAK, WNK insensitive ^T233ASPAK, catalytically inactive ^D212ASPAK, wild-type OSR1, constitutively active ^T185EOSR1, WNK insensitive ^T185AOSR1 and catalytically inactive ^D164AOSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. Results: Coexpression of wild-type SPAK and of ^T233ESPAK, but not of ^T233ASPAK or of ^D212ASPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, ^T185EOSR1 and ^T185AOSR1 but not of ^D164AOSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. Conclusions: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8.

Cloning and characterization of the promoter regions from the parent and paralogous creatine transporter genes

Interconversion between phosphocreatine and creatine, catalyzed by creatine kinase is crucial in the supply of ATP to tissues with high energy demand. Creatine's importance has been established by its use as an ergogenic aid in sport, as well as the development of intellectual disability in patients with congenital creatine deficiency. Creatine biosynthesis is complemented by dietary creatine uptake. Intracellular transport of creatine is carried out by a creatine transporter protein (CT1/CRT/CRTR) encoded by the SLC6A8 gene. Most tissues express this gene, with highest levels detected in skeletal muscle and kidney. There are lower levels of the gene detected in colon, brain, heart, testis and prostate. The mechanism(s) by which this regulation occurs is still poorly understood. A duplicated unprocessed pseudogene of SLC6A8–SLC6A10P has been mapped to chromosome 16p11.2 (contains the entire SLC6A8 gene, plus 2293bp of 5′flanking sequence and its entire 3′UTR). Expression of SLC6A10P has so far only been shown in human testis and brain. It is still unclear as to what is the function of SLC6A10P. In a patient with autism, a chromosomal breakpoint that intersects the 5′flanking region of SLC6A10P was identified; suggesting that SLC6A10P is a non-coding RNA involved in autism. Our aim was to investigate the presence of cis-acting factor(s) that regulate expression of the creatine transporter, as well as to determine if these factors are functionally conserved upstream of the creatine transporter pseudogene.Via gene-specific PCR, cloning and functional luciferase assays we identified a 1104bp sequence proximal to the mRNA start site of the SLC6A8 gene with promoter activity in five cell types. The corresponding 5′flanking sequence (1050bp) on the pseudogene also had promoter activity in all 5 cell lines. Surprisingly the pseudogene promoter was stronger than that of its parent gene in 4 of the cell lines tested. To the best of our knowledge, this is the first experimental evidence of a pseudogene with stronger promoter activity than its parental gene.

A role for thioredoxin-interacting protein (Txnip) in cellular creatine homeostasis

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.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency

BackgroundCreatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and...

214 REGULATION OF CELLULAR CREATINE HOMEOSTASIS: A ROLE FOR THIOREDOXIN INTERACTING PROTEIN (TXNIP)

Background Creatine (Cr) uptake across the plasma membrane is particularly important in excitable cells that cannot synthesise Cr, such as cardiomyocytes, where creatine has a role in energy buffering and transport. Uptake is tightly controlled via a specific transmembrane Creatine Transporter (CrT), while cellular loss is slow, passive, and non-enzymatic. Earlier work has suggested that CrT activity is subject to substrate inhibition, but, that this requires de novo synthesis of unidentified protein(s). The purpose of this study was to identify this protein and to verify its role as a potential endogenous inhibitor of the CrT. Methods and Results To achieve this we took a global gene array approach under varying conditions of cellular [Cr], and verified our findings using an in vitro assay of Cr-uptake using a radiolabelled 14 Cr method. 3T3 fibroblasts stably over-expressing CrT were incubated for 2-5 hours with either low (250 µM) or saturating (5 mM) Cr, with and without cycloheximide (CHX) to inhibit protein synthesis. Cr uptake plateaued at 3 hours in response to saturating levels of extracellular Cr (5mM) and was 33% ( P P =0.036). Quantitative RT-PCR verified up-regulation of Txnip mRNA (61% increase; P=0.002 ) and immunoblotting detected a 45% increase in Txnip protein after 3hr of Cr incubation ( P=0.01 ). Treatment of cells with either 250 µM Cr or 5mM taurine, did not result in Txnip mRNA or protein up-regulation, confirming specificity for saturating Cr. Small interfering RNA against Txnip (siTxnip) reduced mRNA levels by 50% and attenuated the increase in Txnip gene expression in response to 5mM Cr (17% in siTxnip versus 45% in control cells). This was sufficient to maintain CrT activity at control levels during high [Cr], i.e. substrate inhibition was completely abolished. The in vitro observations were tested in vivo in relation to altered left ventricular [Cr]. Cr-deficient mice, due to deletion of the Cr biosynthetic enzyme guanidinoacetate methyl-transferase (GAMT −/− ), had LV Txnip mRNA 35% lower than wild-types. Conversely, mice with cardiac overexpression of CrT had elevated LV [Cr] (mean 124 versus 74nmol/mg protein in WT) associated with a 58% increase in Txnip mRNA and 29% more Txnip protein when compared to wild-type littermates. Conclusion Our work suggests a novel role for Txnip in the endogenous regulation of CrT activity in cardiomyocytes, thereby contributing to modulation of Cr homeostasis. The molecular mechanisms involved in the CrT-Txnip interaction, merit further elucidation.