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Improving energy provision in the failing heart by augmenting the creatine kinase (CK) system is a desirable therapeutic target. However, over-expression of the creatine transporter (CrT-OE) has shown that very high creatine levels result in cardiac hypertrophy and dysfunction. We hypothesise this is due to insufficient endogenous CK activity to maintain thermodynamically favourable metabolite ratios. If correct, then double transgenic mice (dTg) overexpressing both CrT and the muscle isoform of CK (CKM-OE) would rescue the adverse phenotype. In Study 1, overexpressing lines were crossed and cardiac function assessed by invasive haemodynamics and echocardiography. This demonstrated that CKM-OE was safe, but too few hearts had creatine in the toxic range. In Study 2, a novel CrT-OE line was generated with higher, homogeneous, creatine levels and phenotyped as before. Myocardial creatine was 4-fold higher in CrT-OE and dTg hearts compared to wildtype and was associated with hypertrophy and contractile dysfunction. The inability of dTg hearts to rescue this phenotype was attributed to downregulation of CK activity, as occurs in the failing heart. Nevertheless, combining both studies in a linear regression analysis suggests a modest positive effect of CKM over a range of creatine concentrations. In conclusion, we confirm that moderate elevation of creatine is well tolerated, but very high levels are detrimental. Correlation analysis lends support to the theory that this may be a consequence of limited CK activity. Future studies should focus on preventing CKM downregulation to unlock the potential synergy of augmenting both creatine and CK in the heart. [Display omitted] • Improving cardiac energetics via the creatine kinase system can be cardioprotective. • Too much creatine (>2-fold increase) causes heart failure and is not rescued by CK. • LV dysfunction causes transcriptional and post-translational down-regulation of CK. • Aim for modest increases in creatine while preventing loss of CK activity. [ABSTRACT FROM AUTHOR]

Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1β treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis. [ABSTRACT FROM AUTHOR]

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane‐embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148‐creatine‐Na+ is essential to the process of occlusion, which occurs via a "hold‐and‐pull" mechanism. The model should be useful to study the impact of disease‐associated point mutations on the folding of CRT1 and identify approaches which correct folding‐deficient mutants. [ABSTRACT FROM AUTHOR]

Abstract Background Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). Methods and results Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57—mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. Conclusions We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure.

Cancer cells are characterized by uncontrolled growth, proliferation, and impaired apoptosis. Tumour progression could be related to poor prognosis and due to this fact, researchers have been working on novel therapeutic strategies and antineoplastic agents. It is known that altered expression and function of solute carrier proteins from the SLC6 family could be associated with severe diseases, including cancers. These proteins were noticed to play important physiological roles through transferring nutrient amino acids, osmolytes, neurotransmitters, and ions, and many of them are necessary for survival of the cells. Herein, we present the potential role of taurine (SLC6A6) and creatine (SLC6A8) transporters in cancer development as well as therapeutic potential of their inhibitors. Experimental data indicate that overexpression of analyzed proteins could be connected with colon or breast cancers, which are the most common types of cancers. The pool of known inhibitors of these transporters is limited; however, one ligand of SLC6A8 protein is currently tested in the first phase of clinical trials. Therefore, we also highlight structural aspects useful for ligand development. In this review, we discuss SLC6A6 and SLC6A8 transporters as potential biological targets for anticancer agents. [ABSTRACT FROM AUTHOR]

Background: Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). Methods and results: Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57—mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. Conclusions: We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure. [ABSTRACT FROM AUTHOR]

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.

The severe impact on brain function and lack of effective therapy for patients with creatine (Cr) transporter deficiency motivated the generation of three ubiquitous Slc6a8 deficient mice (−/y). While each mouse knock‐out line has similar behavioral effects at 2 to 3 months of age, other features critical to the efficient use of these mice in drug discovery are unclear or lacking: the concentration of Cr in brain and heart differ widely between mouse lines, there are limited data on histopathologic changes, and no data on Cr uptake. Here, we determined survival, measured endogenous Cr and uptake of its deuterium‐labeled analogue Cr‐d3 using a liquid chromatography coupled with tandem mass spectrometry assay, and performed comprehensive histopathologic examination on the Slc6a8−/y mouse developed by Skelton et al. Our results show that Slc6a8−/y mice have widely varying organ‐specific uptake of Cr‐d3, significantly diminished growth with the exception of brain, progressive vacuolar myopathy, and markedly shortened lifespan. [ABSTRACT FROM AUTHOR]

Abstract Inborn errors of metabolism are predominantly autosomal-recessive disorders, but several follow an X-linked pattern of inheritance. They are called X-linked recessive, if the female carriers are asymptomatic, and are called X-linked dominant disorders, if almost all females are affected. Conditions, in which some females have symptoms while others are asymptomatic lifelong are simply referred to as X-linked. The aim of this review is to point out the variability in clinical manifestation of affected females in some X-linked metabolic disorders and to discuss on the basis of these examples possible mechanisms that may explain the broad phenotypic spectrum, such as the type of the underlying mutation, the issue of autonomous versus non-autonomous gene expression and the degree of skewing of X-inactivation. The use of the terms “X-linked dominant” and “X-linked recessive” will be discussed.

Purpose: Cyclocreatine, a creatine analog, is a candidate drug for treating patients with cerebral creatine deficiency syndromes (CCDSs) caused by creatine transporter (CRT, SLC6A8) deficiency, which reduces brain creatine level. The purpose of this study was to clarify the characteristics of cyclocreatine transport in HEK293 cells, which highly express endogenous CRT, in hCMEC/D3 cells, a human blood-brain barrier (BBB) model, and in CCDSs patient-derived fibroblasts with CRT mutations. Methods: Cells were incubated at 37°C with [14C]cyclocreatine (9 μM) and [14C]creatine (9 μM) for specified periods of times in the presence or absence of inhibitors, while the siRNAs were transfected by lipofection. Protein expression and mRNA expression were quantified using targeted proteomics and quantitative PCR, respectively. Results: [14C]Cyclocreatine was taken up by HEK293 cells in a time-dependent manner, while exhibiting saturable kinetics. The inhibition and siRNA knockdown studies demonstrated that the uptake of [14C]cyclocreatine by both HEK293 and hCMEC/D3 cells was mediated predominantly by CRT as well as [14C]creatine. In addition, uptake of [14C]cyclocreatine and [14C]creatine by the CCDSs patient-derived fibroblasts was found to be largely reduced. Conclusion: The present study suggests that cyclocreatine is a CRT substrate, where CRT is the predominant contributor to influx of cyclocreatine into the brain at the BBB. Our findings provide vital insights for the purposes of treating CCDSs patients using cyclocreatine. [ABSTRACT FROM AUTHOR]

The lack of cerebral creatine (Cr) causes intellectual disability and epilepsy. In addition, a significant portion of individuals with Cr transporter (Crt) deficiency (CTD), the leading cause of cerebral Cr deficiency syndromes (CCDS), are diagnosed with attention-deficit hyperactivity disorder. While the neurological effects of CTD are clear, the mechanisms that underlie these deficits are unknown. Part of this is due to the heterogenous nature of the brain and the unique metabolic demands of specific neuronal systems. Of particular interest related to Cr physiology are dopaminergic neurons, as many CCDS patients have ADHD and Cr has been implicated in dopamine-associated neurodegenerative disorders, such as Parkinson's and Huntington's diseases. The purpose of this study was to examine the effect of a loss of the Slc6a8 (Crt) gene in dopamine transporter (Slc6a3; DAT) expressing cells on locomotor activity and motor function as the mice age. Floxed Slc6a8 (Slc6a8flox) mice were mated to DATIREScre expressing mice to generate DAT-specific Slc6a8 knockouts (dCrt−/y). Locomotor activity, spontaneous activity, and performance in the challenging beam test were evaluated monthly in dCrt−/y and control (Slc6a8flox) mice from 3 to 12 months of age. dCrt−/y mice were hyperactive compared with controls throughout testing. In addition, dCrt−/y mice showed increased rearing and hindlimb steps in the spontaneous activity test. Latency to cross the narrow bridge was increased in dCrt−/y mice while foot slips were unchanged. Taken together, these data suggest that the lack of Cr in dopaminergic neurons causes hyperactivity while sparing motor function. [ABSTRACT FROM AUTHOR]

[Display omitted] • We used Gad2-Cre expressing mice to drive a GABA-specific deletion of the creatine transporter (Slc6a8) gene. • Gad2-Cre specific Slc6a8 knockout mice (cKO) had contextual and cued freezing deficits similar to Slc6a8 knockout mice. • Spatial learning and memory were spared in cKO mice. • Gad2-Cre+ mice had object recognition memory deficits; confounding deficits seen in the cKO mice. The creatine (Cr)-phosphocreatine shuttle is essential for ATP homeostasis. In humans, the absence of brain Cr causes significant intellectual disability, epilepsy, and language delay. Mutations of the creatine transporter (SLC6A8) are the most common cause of Cr deficiency. In rodents, Slc6a8 deletion causes deficits in spatial learning, novel object recognition (NOR), as well as in contextual and cued freezing. The mechanisms that underlie these cognitive deficits are not known. Due to the heterogeneous nature of the brain, it is important to determine which systems are affected by a loss of Cr. In this study, we generated mice lacking Slc6a8 in GABAergic neurons by crossing Slc6a8FL mice with Gad2-Cre mice. These Gad2-specific Slc6a8 knockout (cKO) mice, along with the ubiquitous Slc6a8 KO (Slc6a8-/y), Gad2-Cre+ , and wild-type (WT) mice were tested in the Morris water maze, NOR, conditioned freezing, and the radial water maze. Similar to the Slc6a8-/y mice, cKO mice had reduced contextual and cued freezing compared with WT mice. The cKO mice had a mild spatial learning deficit during the reversal phase of the MWM, however they were not as pronounced as in Slc6a8-/y mice. In NOR, the Gad2-Cre mice spent less time with the novel object, similar to the reduced novel time in the cKO mice. There were no changes in radial water maze performance. Slc6a8 deletion in GABAergic neurons is sufficient to recapitulate the conditioned freezing deficits seen in Slc6a8-/y mice. [ABSTRACT FROM AUTHOR]

[Display omitted] • Collection and analysis of 30 missense variants of the human creatine transporter (SLC6A8). • Study of transport activity, subcellular localization, and in silico modeling of SLC6A8 variants. • SLC6A8 interactome and computational modeling of SLC6A8 protein–protein interactions. • Variant effect disambiguation of 15 SLC6A8 variants of unknown effect. Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter's conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome. [ABSTRACT FROM AUTHOR]

This study examined the effect of vegetarianism on skeletal muscle total creatine (TCr) content and creatine transporter (CreaT) gene expression, prior to and during 5 d of Cr supplementation (CrS). In a double-blind, crossover design, 7 vegetarians (VEG) and nonvegetarians (NVEG) were assigned Cr or placebo supplements for 5 d and after 5 wk, received the alternative treatment. Muscle sampling occurred before, and after 1 and 5 d of treatment ingestion. Basal muscle TCr content was lower (P < 0.05) in VEG compared with NVEG. Muscle TCr increased (P < 0.05) throughout the Cr trial in both groups but was greater (P < 0.05) in VEG compared with NVEG, at days 1 and 5. CreaT gene expression was not different between VEG and NVEG. The results indicate that VEG have a lower muscle TCr content and an increased capacity to load Cr into muscle following CrS. Muscle CreaT gene expression does not appear to be affected by vegetarianism. [ABSTRACT FROM AUTHOR]

Background: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium. Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes, co-expression of GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt. Conclusion: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt.

Cerebral creatine deficiency syndromes (CCDS) are neurodevelopmental disorders caused by a decrease in creatine levels in the central nervous system (CNS) due to functional mutations in creatine synthetic enzymes or creatine transporter (CRT/SLC6A8). Although SLC6A8 mutations have been reported to be the most frequent cause of CCDS, sufficient treatment for patients with CCDS harboring SLC6A8 mutations has not yet been achieved. This study aimed to elucidate the molecular mechanism of SLC6A8 dysfunction caused by the c. 1699T > C missense mutation, which is thought to induce dysfunction through an unidentified mechanism. A study on SLC6A8-expressing oocytes showed that the c.1699T > C mutation decreased creatine uptake compared to that in wild-type (WT) oocytes. In addition, a kinetics study of creatine uptake revealed that the c.1699T > C mutation reduced the maximum uptake rate but not Michaelis-Menten constant. In contrast, the c.1699T > C mutation did not attenuate SLC6A8 protein levels or alter its cellular localization. Based on the SLC6A8 structure in the AlphaFold protein structure database, it is possible that the c.1699T > C mutation alters the interaction between the S567 and Y143 residues of SLC6A8, leading to decreased creatine transport function. These findings contribute to the understanding of the pathology of CCDS and to the development of strategies for CCDS treatment.

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane-embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148-creatine-Na + is essential to the process of occlusion, which occurs via a "hold-and-pull" mechanism. The model should be useful to study the impact of disease-associated point mutations on the folding of CRT1 and identify approaches which correct folding-deficient mutants., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Creatine serves as fast energy buffer in organs of high-energy demand such as brain and skeletal muscle. L-Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase are responsible for endogenous creatine synthesis. Subsequent uptake into target organs like skeletal muscle, heart and brain is mediated by the creatine transporter (CT1, SLC6A8). Creatine deficiency syndromes are caused by defects of endogenous creatine synthesis or transport and are mainly characterized by intellectual disability, behavioral abnormalities, poorly developed muscle mass, and in some cases also muscle weakness. CT1-deficiency is estimated to be among the most common causes of X-linked intellectual disability and therefore the brain phenotype was the main focus of recent research. Unfortunately, very limited data concerning muscle creatine levels and functions are available from patients with CT1 deficiency. Furthermore, different CT1-deficient mouse models yielded conflicting results and detailed analyses of their muscular phenotype are lacking. Here, we report the generation of a novel CT1-deficient mouse model and characterized the effects of creatine depletion in skeletal muscle. HPLC-analysis showed strongly reduced total creatine levels in skeletal muscle and heart. MR-spectroscopy revealed an almost complete absence of phosphocreatine in skeletal muscle. Increased AGAT expression in skeletal muscle was not sufficient to compensate for insufficient creatine transport. CT1-deficient mice displayed profound impairment of skeletal muscle function and morphology (i.e., reduced strength, reduced endurance, and muscle atrophy). Furthermore, severely altered energy homeostasis was evident on magnetic resonance spectroscopy. Strongly reduced phosphocreatine resulted in decreased ATP/Pi levels despite an increased inorganic phosphate to ATP flux. Concerning glucose metabolism, we show increased glucose transporter type 4 expression in muscle and improved glucose clearance in CT1-deficient mice. These metabolic changes were associated with activation of AMP-activated protein kinase – a central regulator of energy homeostasis. In summary, creatine transporter deficiency resulted in a severe muscle weakness and atrophy despite different compensatory mechanisms.

Creatine transporter (CrT; SLC6A8) deficiency (CTD) is an X‐linked disorder characterized by severe cognitive deficits, impairments in language and an absence of brain creatine (Cr). In a previous study, we generated floxed Slc6a8 (Slc6a8 flox) mice to create ubiquitous Slc6a8 knockout (Slc6a8−/y) mice. Slc6a8−/y mice lacked whole body Cr and exhibited cognitive deficits. While Slc6a8−/y mice have a similar biochemical phenotype to CTD patients, they also showed a reduction in size and reductions in swim speed that may have contributed to the observed deficits. To address this, we created brain‐specific Slc6a8 knockout (bKO) mice by crossing Slc6a8flox mice with Nestin‐cre mice. bKO mice had reduced cerebral Cr levels while maintaining normal Cr levels in peripheral tissue. Interestingly, brain concentrations of the Cr synthesis precursor guanidinoacetic acid were increased in bKO mice. bKO mice had longer latencies and path lengths in the Morris water maze, without reductions in swim speed. In accordance with data from Slc6a8 −/y mice, bKO mice showed deficits in novel object recognition as well as contextual and cued fear conditioning. bKO mice were also hyperactive, in contrast with data from the Slc6a8 −/y mice. The results show that the loss of cerebral Cr is responsible for the learning and memory deficits seen in ubiquitous Slc6a8−/y mice. [ABSTRACT FROM AUTHOR]

Creatine serves as fast energy buffer in organs of high-energy demand such as brain and skeletal muscle. L-Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase are responsible for endogenous creatine synthesis. Subsequent uptake into target organs like skeletal muscle, heart and brain is mediated by the creatine transporter (CT1, SLC6A8). Creatine deficiency syndromes are caused by defects of endogenous creatine synthesis or transport and are mainly characterized by intellectual disability, behavioral abnormalities, poorly developed muscle mass, and in some cases also muscle weakness. CT1-deficiency is estimated to be among the most common causes of X-linked intellectual disability and therefore the brain phenotype was the main focus of recent research. Unfortunately, very limited data concerning muscle creatine levels and functions are available from patients with CT1 deficiency. Furthermore, different CT1-deficient mouse models yielded conflicting results and detailed analyses of their muscular phenotype are lacking. Here, we report the generation of a novel CT1-deficient mouse model and characterized the effects of creatine depletion in skeletal muscle. HPLC-analysis showed strongly reduced total creatine levels in skeletal muscle and heart. MR-spectroscopy revealed an almost complete absence of phosphocreatine in skeletal muscle. Increased AGAT expression in skeletal muscle was not sufficient to compensate for insufficient creatine transport. CT1-deficient mice displayed profound impairment of skeletal muscle function and morphology (i.e., reduced strength, reduced endurance, and muscle atrophy). Furthermore, severely altered energy homeostasis was evident on magnetic resonance spectroscopy. Strongly reduced phosphocreatine resulted in decreased ATP/Pi levels despite an increased inorganic phosphate to ATP flux. Concerning glucose metabolism, we show increased glucose transporter type 4 expression in muscle and improved glucose clearance in CT1-deficient mice. These metabolic changes were associated with activation of AMP-activated protein kinase - a central regulator of energy homeostasis. In summary, creatine transporter deficiency resulted in a severe muscle weakness and atrophy despite different compensatory mechanisms. [ABSTRACT FROM AUTHOR]

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 ( D 3 -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. [ABSTRACT FROM AUTHOR]

Background: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium. Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes, co-expression of GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt. Conclusion: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt. [ABSTRACT FROM AUTHOR]

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose ( r² = 0.45; P = 0.002), acetyl-carnitine ( r² = 0.50; P = 0.001), glutamine ( r² = 0.59; P = 0.0002); and negative correlations with taurine ( r² = 0.74; P < 0.0001), fumarate ( r² = 0.45; P = 0.003), aspartate ( r² = 0.59; P = 0.0002), alanine ( r² = 0.66; P < 0.0001) and phosphocholine ( r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels. [ABSTRACT FROM AUTHOR]

Creatine (Cr) is a guanidino compound required for rapid replenishment of ATP in cells with a high-energy demand. In humans, mutations in the Cr transporter ( CRT; SLC6A8) prevent Cr entry into tissue and result in a significant intellectual impairment, epilepsy, and aphasia. The lack of Cr on both the whole body and cellular metabolism was evaluated in Crt knockout ( Crt ) mice, a high-fidelity model of human CRT deficiency. Crt mice have reduced body mass and, however, show a twofold increase in body fat. There was increased energy expenditure in a home cage environment and during treadmill running in Crt mice. Consistent with the increases in the whole-body metabolic function, Crt mice show increased cellular metabolism as well. Mitochondrial respiration increased in skeletal muscle fibers and hippocampal lysates from Crt mice. In addition, Crt mice had increased citrate synthase activity, suggesting a higher number of mitochondria instead of an increase in mitochondrial activity. To determine if the increase in respiration was due to increased mitochondrial numbers, we measured oxygen consumption in an equal number of mitochondria from Crt and Crt mice. There were no changes in mitochondrial respiration when normalized to mitochondrial number, suggesting that the increase in respiration observed could be to higher mitochondrial content in Crt mice. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT-/y) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT-/y and wild type (CrT+/y) mice were analysed for ATP, Cr, Cr phosphate (CrP) and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT-/y mice muscle contained measurable (22.3 ± 4.3 mmol.kg-1 dry mass), but markedly reduced (P

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. [ABSTRACT FROM AUTHOR]

A confined absorption of exogenous creatine through creatine transporter (CRT1) seems to hamper its optimal uptake in bioenergetical deficits. Co-administration of guanidinoacetic acid (GAA) along with creatine could target other transport channels besides CRT1, and supremely improve cellular levels of creatine. This innovative approach might tackle tissues difficult to reach with conventional creatine interventions, providing a potentially more effective and safe mixture in clinical pharmacology and therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Background: Aberrations 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. Methods: Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport. Results: SLC6A8D 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. Conclusions: CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake. General significance: Provides added insight into the mechanism(s) of creatine transport regulation. [Copyright &y& Elsevier]

Creatine and phosphocreatine levels are decreased in heart failure, and reductions in myocellular phosphocreatine levels predict the severity of the disease and portend adverse outcomes. Previous studies of transgenic mouse models with increased creatine content higher than two times baseline showed the development of heart failure and shortened lifespan. Given phosphocreatine's role in buffering ATP content, we tested the hypothesis whether elevated cardiac creatine content would alter cardiac function under normal physiological conditions. Here, we report the creation of transgenic mice that overexpress the human creatine transporter (CrT) in cardiac muscle under the control of the myosin heavy chain promoter. Cardiac transgene expression was quantified by qRT-PCR, and human CrT protein expression was documented on Western blots and immunohistochemistry using a specific anti-CrT antibody. High-energy phosphate metabolites and cardiac function were measured in transgenic animals and compared with age-matched, wild-type controls. Adult transgenic animals showed increases of 5.7- and 4.7-fold in the content of creatine and free ADP, respectively. Phosphocreatine and ATP levels were two times as high in young transgenic animals but declined to control levels by the time the animals reached 8 wk of age. Transgenic mice appeared to be healthy and had normal life spans. Cardiac morphometry, conscious echocardiography, and pressure-volume loop studies demonstrated mild hypertrophy but normal function. Based on our characterization of the human CrT protein expression, creatine and phosphocreatine content, and cardiac morphometry and function, these transgenic mice provide an in vivo model for examining the therapeutic value of elevated creatine content for cardiac pathologies. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lygate, Lake, McAndrew, Neubauer and Zervou.)

The article presents a study on the tissue distribution of porcine creatine transporter from Landrace pigs. The investigation was done by cloning the complementary DNA (cDNA) of porcine skeletal muscle creatine transporter (CrT) with the use of rapid amplification of cDNA end technology. It asserts that the procedure will benefit for a better understanding of the creatine metabolism in pigs and help in the field of meat quality.

Abstract: Creatine transporter deficiency is an X-linked disorder caused by mutations in the SLC6A8 gene. Currently, 38 pathogenic, including 15 missense variants, are reported. In this study, we report 33 novel, including 6 missense variants. To classify all known missense variants, we transfected creatine deficient fibroblasts with the SLC6A8 ORF containing one of the unique variants and tested their ability to restore creatine uptake. This resulted in the definitive classification of 2 non-disease associated and 19 pathogenic variants of which 3 have residual activity. Furthermore, we report the development and validation of a novel DHPLC method for the detection of heterozygous SLC6A8 variants. The method was validated by analysis of DNAs that in total contained 67 unique variants of which 66 could be detected. Therefore, this rapid screening method may prove valuable for the analysis of large cohorts of females with mild intellectual disability of unknown etiology, since in this group heterozygous SLC6A8 mutations may be detected. DHPLC proved also to be important for the detection of somatic mosaicism in mothers of patients who have a pathogenic mutation in SLC6A8. All variants reported in the present and previous studies are included in the Leiden Open Source Variant Database (LOVD) of SLC6A8 (www.LOVD.nl/SLC6A8). [Copyright &y& Elsevier]

J. Neurochem. (2010) 115, 684-693. Creatine uptake by neurons requires a specific creatine transporter (CRT). The purpose of the present work was to investigate the activity and localization of the CRT in primary cultures of hippocampal neurons obtained from 18-day rat embryos. Creatine uptake increased as the neurons differentiated in culture. Immunofluorescence microscopy showed most of the CRT was associated with dendrites, although some CRT was present in axons and axon terminals. Neurons contained high levels of Na+-dependent creatine transport activity ( Km = 45.5 μM; Vmax, 1719 pmol creatine/min/mg protein) which was inhibited by competitive inhibitors of the CRT. The IC50 for guanidinoacetate, a precursor of creatine, was 712 μM, ∼ 15-fold higher than the Km for creatine. Incubation of neurons with 1 mM creatine resulted in the accumulation of high levels of creatine which affected the Vmax but not the Km for creatine transport. The rate of creatine release from neurons increased in the absence of Na+ showing the importance of the electrochemical gradient for creatine retention. This is the first detailed study of the CRT in neurons and identifies primary cultures of rat hippocampal neurons as a good model for future studies of the CRT in relation to the effects of creatine on neuronal function and viability. [ABSTRACT FROM AUTHOR]

X-linked creatine transporter defect is caused by mutations in SLC6A8 at Xq28, which encodes the sodium-dependent creatine transporter. Reduction in creatine uptake results in elevated urine creatine and CSF creatine deficiency, which can be detected on magnetic resonance spectroscopy. We report a patient who was initially suspected of having a mitochondrial disorder but was later found to have a creatine transporter defect. The abnormal laboratory study results seen in this patient suggesting a mitochondrial cytopathy could be due to excess mitochondrial stress as well as the mitochondrial inclusion bodies. This report looks at the mitochondrial presentation of the creatine transporter deficiency. [ABSTRACT FROM AUTHOR]

In cells and tissues with a high demand of energy such as neurons the major pool of energy is supplied by phosphocreatine. The necessary supply with creatine is directly related to the expression level of the creatine transporter (CRT). To study possible interactions between this transporter and depressive like behavior we have used a congenitally learned helplessness (cLH) model. Because uptake via CRT is the only means to supply neurons with creatine, we hypothesized a rise in CRT expression following antidepressive treatment strategies. The hippocampus of the cLH animals exhibited a significantly lower CRT expression as compared to wild type (WT) animals. Treatment with escitalopram and ECS induced an elevation of CRT levels in the hippocampus and the prefrontal cortex. In WT animals there was no treatment effect. In summary, our data presented here suggest an association between depressive behavior and cellular energy metabolism in defined brain areas. [ABSTRACT FROM AUTHOR]

Although the cerebral accumulation of guanidinoacetate (GAA) contributes to neurological complications in S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) deficiency, how GAA is abnormally distributed in the brain remains unknown. The purpose of this study was to investigate the transport of GAA across the blood–brain barrier (BBB) and in brain parenchymal cells in rats. [14C]GAA microinjected into the rat cerebrum was not eliminated from the brain, implying the negligible contribution of GAA efflux transport across the BBB. In contrast, in vivo analysis and an uptake study by TR-BBB cells, a rat in vitro BBB model, revealed that GAA was transported from the circulating blood across the BBB most likely via a creatine transporter (CRT). Although CRT at the BBB is almost saturated by endogenous creatine under physiological conditions, the creatine level in the blood significantly decreases in GAMT deficiency. This might lead to the increase of CRT-mediated blood-to-brain transport of GAA at the BBB. Furthermore, [14C]GAA was taken up by brain parenchymal cells in a concentrative manner most likely via taurine transporter and CRT. These characteristics of GAA transport across the BBB and in the brain parenchymal cells could be the key factors that facilitate GAA accumulation in the brains of patients with GAMT deficiency. [ABSTRACT FROM AUTHOR]

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a Km value of 269 μM/412 μM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that ofd-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB. [ABSTRACT FROM AUTHOR]

There is still incomplete evidence for the cerebral clearance of creatinine (CTN) which is an endogenous convulsant and accumulates in the brain and CSF of patients with renal failure. The purpose of this study was to clarify the transporter-mediated CTN efflux transport from the brain/CSF. In vivo data demonstrated that CTN after intracerebral administration was not significantly eliminated from the brain across the blood-brain barrier. In contrast, the elimination clearance of CTN from the CSF was 60-fold greater than that of inulin, reflecting CSF bulk flow. Even in renal failure model rats, the increasing ratio of the CTN concentration in the CSF was lower than that in the plasma, suggesting a significant role for the CSF-to-blood efflux process. The inhibitory effects of inhibitors and antisense oligonucleotides on CTN uptake by isolated choroid plexus indicated the involvement of rat organic cation transporter 3 (rOCT3) and creatine transporter (CRT) in CTN transport. rOCT3- and CRT-mediated low-affinity CTN transport with Km values of 47.7 and 52.0 mM, respectively. Our findings suggest that CTN is eliminated from the CSF across the blood-CSF barrier as a major pathway of cerebral CTN clearance and transporter-mediated processes are involved in the CTN transport in the choroid plexus. [ABSTRACT FROM AUTHOR]

Abstract: Creatine (Cr) is an amino acid, which upon phosphorylation is utilized as an energy reservoir in cells with high-energy demand. The ongoing catabolism of creatine to creatinine requires a permanent creatine replenishment into the cells. Because neurons themselves cannot synthesize creatine, they have to take it up via the creatine transporter (CrT). Thus, the concentration of intracellular Cr available for the Cr/PCr shuttle system depends on the expression level of CrT protein. The proton magnetic resonance spectroscopy (MRS) creatine peak (total creatine=tCr) constitutes of two metabolites, namely Cr and phosphocreatine (PCr). We have quantified the level of CrT protein expression with western blotting and compared it to tCr content as estimated by in vitro MRS in Sprague–Dawley rats. Under the assumption of hemispheric symmetry, we took identical samples from left and right hemisphere, which were used for in vitro MRS (tCr) and for western blotting (CrT), respectively. Altogether, it was possible to take 90 corresponding brain samples from 31 animals. A Pearson linear regression analysis for CrT and tCr revealed p <0.0001, explaining 14% of the variance. Since MR-detectable alterations of tCr in the human brain are widespread (e.g. in most major psychiatric disorders proton MRS detectable tCr alterations have been described as regionally and usually state dependent) it is stringent to elucidate their meaning. An influence of tCr on the brain''s energy regulating system seems plausible. [Copyright &y& Elsevier]

Abstract: Characteristic alterations of the creatine kinase (CK) system occur in heart failure and may contribute to contractile dysfunction. We examined two mouse models of chronic cardiac stress, transverse aortic constriction (TAC) and coronary artery ligation (CAL), and examined the relationship of CK system changes with hypertrophy and heart failure development. C57Bl/6 mice were subjected to TAC or sham surgery and sacrificed after 2–10 weeks according to echocardiographic criteria of myocardial hypertrophy and function to create four groups representing progressive dysfunction from normal, through compensated hypertrophy, to heart failure. Only mice with congestive heart failure had LV total creatine concentration and total CK activity significantly lower than sham values (11% and 30% lower, respectively). However for all aortic banded mice, a linear relationship was observed between ejection fraction and estimated maximal CK reaction velocity. Mice with heart failure also had corresponding decreases in the activities of the Mito-, MM-, and MB-CK isoenzymes, while the BB isoform remained unchanged. To determine whether these changes were model specific, mice were subjected to CAL or sham operation and followed for 7 weeks. Quantitative changes in total creatine, total CK activity, Mito-CK and MM-CK activities were similar for CAL and TAC mice. We conclude that alterations in the creatine kinase system occur during heart failure in mice qualitatively similar to those occurring in larger animals and humans, suggesting that mice are a suitable model for studying the role of such changes in the pathogenesis of heart failure. [Copyright &y& Elsevier]

Creatine (Cr) levels in skeletal muscle and brain of a mouse model of Cr deficiency caused by guanidinoacetate methyltransferase absence (GAMT-/-) were studied after Cr supplementation with 2 g·kg body wt-1·day-1 Cr for 35 days. Localized ¹H magnetic resonance spectroscopy (MRS) was performed in brain (cerebellum and thalamus/hippocampus) and in hind leg muscle of GAMT-/- mice before and after Cr supplementation and in control (Con) mice. As expected, a signal for Cr was hardly detectable in MR spectra of GAMT-/- mice before Cr supplementation. In the thalamus/hippocampus region of these mice, an increase in N-acetylasparate (NAA) was observed. During Cr administration, Cr levels increased faster in skeletal muscle compared with brain, but this occurred only during the first day of supplementation. Thereafter, Cr levels increased by 0.8 mM/day in all studied locations. After 35 days of Cr supplementation, Cr levels in all locations were higher compared with Con mice on a Cr-free diet and NAA levels normalized. Only because of the repeated MRS measurements performed in this longitudinal Cr supplementation study on GAMT-/- mice were we able to discover the initial faster uptake of Cr in skeletal muscle compared with brain, which may represent muscular Cr uptake independent of Cr transporter expression. Our results can provide the basis for additional experiments to optimize Cr supplementation in GAMT deficiency, as increases in brain Cr are slow in patients after Cr supplementation. [ABSTRACT FROM AUTHOR]

Abstract: Ammonia is a key neurotoxin involved in the neurological complications of acute liver failure. The present study was undertaken to study the effects of exposure to pathophysiologically relevant concentrations of ammonium chloride on cultured brain capillary endothelial cells in order to identify mechanisms by which ammonia may alter blood–brain barrier function. Conditionally immortalized mouse brain capillary endothelial cells (TM-BBB) were used as an in vitro model of the blood–brain barrier. Gene expression of a series of blood–brain barrier transporters and tight junction proteins was assessed by quantitative real time PCR analysis. Exposure to ammonia (5mM for 72h) resulted in significant increases in mRNA levels of taurine transporter (TAUT; 2.0-fold increase) as well as creatine transporter (CRT; 1.9-fold increase) whereas claudin-12 mRNA expression was significantly reduced to 67.7% of control levels. Furthermore, [3H]taurine and [14C]creatine uptake were concomitantly increased following exposure to ammonia, suggesting that up-regulation of both TAUT and CRT under hyperammonemic conditions results in an increased function of these two transporters in TM-BBB cells. TAUT and CRT are respectively involved in osmoregulation and energy buffering in the brain, two systems that are thought to be affected in acute liver failure. Furthermore, claudin-12 down-regulation suggests that hyperammonemia may also affect tight junction integrity. Our results provide evidence that ammonia can alter brain capillary endothelial cell gene expression and transporter function. These findings may be relevant to pathological situations involving hyperammonemia, such as liver disease. [Copyright &y& Elsevier]

Abstract: Cobaltic protoporphyrin IX (CoPP) is a synthetic heme analog which can elicit profound and prolonged decreases in appetite and body weight in several different animal species. Intracerebroventricular administration of CoPP in rats was found, by differential display and confirmed by Real-Time PCR, to result in an increase in expression of the creatine transporter when compared to vehicle-treated fed or vehicle-treated fasted control animals. In situ hybridization studies showed that creatine transporter mRNA concentrations were increased in several areas of the brain involved in the regulation of food intake, but creatine concentrations were decreased in hypothalamic homogenates in CoPP-treated animals compared to controls. Intracerebroventricular administration of β-guanidinopropionic acid, a compound known to decrease intracellular creatine concentration by competition for uptake, resulted in decreased food intake and body weight and increased Fos expression in the hypothalamus. Taken together, these findings suggest that creatine concentrations in the brain may play a role in regulating food intake and body weight. [Copyright &y& Elsevier]

Mutations in the creatine transporter gene, SLC6A8 (MIM 30036), located in Xq28, have been found in families with X-linked mental retardation (XLMR) as well as in males with idiopathic mental retardation (MR). In order to estimate the frequency of such mutations in the MR population, a screening of 478 males with MR of unknown cause was undertaken. All 13 exons of SLC6A8 were sequenced using genomic DNA. Six novel potentially pathogenic mutations were identified that were not encountered in at least 588 male control chromosomes: two deletions (p.Asn336del, p.Ile347del) and a splice site alteration (c.1016+2C>T) are considered pathogenic based on the nature of the variant. A mutation (p.Arg391Trp) should be considered pathogenic owing to its localization in a highly conserved region. Two other missense variants (p.Lys4Arg, p.Gly26Arg) are not conserved but were not observed in over 300 male control chromosomes. Their pathogenicity is uncertain. A missense variant (p.Val182Met), was classified as a polymorphism based on a normal creatine/creatinine (Cr:Crn) ratio and cerebral creatine signal in proton magnetic resonance spectroscopy (H-MRS) in the patient. Furthermore, we found 14 novel intronic and neutral variants that were not encountered in at least 280 male control chromosomes and should be considered as unclassified variants. Our findings of a minimum of four pathogenic mutations and two potentially pathogenic mutations indicate that about 1% of males with MR of unknown etiology might have a SLC6A8 mutation. Thus, DNA sequence analysis and/or a Cr:Crn urine screen is warranted in any male with MR of unknown cause. [ABSTRACT FROM AUTHOR]

The diagnosis of creatine deficiencies, a paediatric application of magnetic resonance spectroscopy that has already become a diagnostic tool in clinical practice, is reviewed and illustrated with results from recent examinations. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, three inherited defects in the biosynthesis and transport of creatine have been described. The biosynthetic defects include deficiencies of l-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. The third defect is a functional defect in the creatine transporter (SLC6A8). Clinical symptoms of the three defects vary in severity, are aspecific and include mental retardation with severe speech delay, autistiform behaviour, and epilepsy. Some patients with GAMT deficiency exhibit a more complex clinical phenotype with extrapyramidal movement disorder. All three defects can be diagnosed by in vivo proton magnetic resonance spectroscopy of the brain, which shows a severe reduction or absence of creatine. Laboratory investigations for the diagnosis start with the analysis of guanidinoacetate, creatine and creatinine in body fluids (plasma and urine). Based on these findings, enzyme assays for AGAT or GAMT, or a creatine uptake assay for the transporter defect can be performed. DNA mutation analysis of the genes involved can prove the defects at the molecular level. To diagnose female patients with SLC6A8 deficiency, mutation analysis may be the only choice. [Copyright &y& Elsevier]

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. [ABSTRACT FROM AUTHOR]