Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

Homoarginine levels are regulated by L-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies.

BACKGROUND: Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. METHODS AND RESULTS: Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64-0.96]; P=0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke (P<0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the L-arginine:glycine amidinotransferase (AGAT) gene (P<2.1 × 10(-8); n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT(-/-)) and (2) a guanidinoacetate N-methyltransferase deletion (GAMT(-/-)) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT(-/-) mice and increased in GAMT(-/-) mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT(-/-) mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT(-/-) mice was decreased compared with controls. CONCLUSIONS: Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Creatine deficiency syndrome. A treatable myopathy due to arginine-glycine amidinotransferase (AGAT) deficiency.

We report two sisters, aged 11 and 6years, with AGAT deficiency syndrome (OMIM 612718) which is the least common creatine deficiency syndrome. They were born full-term to consanguineous parents and had moderate developmental delay. Examination showed an important language delay, a progressive proximal muscular weakness in the lower limbs with Gowers sign and myopathic electromyography. Investigations revealed undetectable guanidinoacetate and low level of creatine in plasma and urine, characteristic findings of AGAT deficiency syndrome. Brain magnetic resonance spectroscopy showed a markedly reduced level of creatine. Guanidinoacetate methyltransferase (GATM) gene sequencing revealed a homozygous missense mutation in exon 4:c.608A>C, (p.Tyr203Ser). Thirteen months after beginning the treatment with oral creatine monohydrate 200mg/kg/day, then 400mg/kg/day, there was a dramatic improvement in muscle strength with Gowers sign disappearance in both patients, and a mild improvement in language and cognitive functions. AGAT deficiency syndrome should be considered in all patients with language retardation and cognitive impairment associated to a myopathy of unknown etiology such that early diagnosis must lead to creatine supplementation to cure the myopathy and improve language and cognitive function.

Maternal dietary creatine supplementation does not alter the capacity for creatine synthesis in the newborn spiny mouse.

We have previously reported that maternal creatine supplementation protects the neonate from hypoxic injury. Here, we investigated whether maternal creatine supplementation altered expression of the creatine synthesis enzymes (arginine:glycine amidinotransferase [AGAT], guanidinoaceteate methyltransferase [GAMT]) and the creatine transporter (solute carrier family 6 [neurotransmitter transporter, creatine] member 8: SLC6A8) in the term offspring. Pregnant spiny mice were fed a 5% creatine monohydrate diet from midgestation (day 20) to term (39 days). Placentas and neonatal kidney, liver, heart, and brain collected at 24 hours of age underwent quantitative polymerase chain reaction and Western blot analysis. Maternal creatine had no effect on the expression of AGAT and GAMT in neonatal kidney and liver, but mRNA expression of AGAT in brain tissues was significantly decreased in both male and female neonates born to mothers who were fed the creatine diet. SLC6A8 expression was not affected by maternal dietary creatine loading in any tissues. Maternal dietary creatine supplementation from midgestation in the spiny mouse did not alter the capacity for creatine synthesis or transport.

L-arginine:glycine amidinotransferase deficiency protects from metabolic syndrome.

Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.

Developmental progress and creatine restoration upon long-term creatine supplementation of a patient with arginine:glycine amidinotransferase deficiency.

BACKGROUND: Arginine:glycineamidinotransferase (AGAT/GATM) deficiency has been described in 9 patients across 4 families. Here we describe the clinical outcome and response to creatine supplementation in a patient of the second family affected with AGAT deficiency-a 9-year-old girl. PATIENT AND METHODS: Delayed motor milestones were noticed from 4 months of age and at 14 months moderate hypotonia, developmental delay and failure to thrive. Laboratory studies revealed low plasma creatine as well as extremely low levels of guanidinoacetic acid in urine and plasma. Proton magnetic resonance spectroscopy (MRS) of the brain showed absence of creatine. DNA sequence analysis revealed a homozygous mutation (c.484+1G>T) in the AGAT/GATM gene. AGAT activity was not detectable in lymphoblasts and RNA analysis revealed a truncated mRNA (r.289_484del196) that is degraded via Nonsense Mediated Decay. At 16 months, Bayley's Infant Development Scale (BIDS) showed functioning at 43% of chronologic age. Oral creatine supplementation (up to 800 mg/kg/day) was begun. RESULTS: At age 9 years she demonstrated advanced academic performance. Partial recovery of cerebral creatine levels was demonstrated on MRS at 25 months of age. Brain MRS at 40 months of age revealed a creatine/NAA ratio of about 80% of that in age-matched controls. CONCLUSIONS: 8 years post initiation of oral creatine supplementation, patient demonstrates superior nonverbal and academic abilities, with average verbal skills. We emphasize that early diagnosis combined with early treatment onset of AGAT deficiency may lead to improvement of developmental outcome.

Disorders of creatine transport and metabolism.

Creatine is a nitrogen containing compound that serves as an energy shuttle between the mitochondrial sites of ATP production and the cytosol where ATP is utilized. There are two known disorders of creatine synthesis (both transmitted as autosomal recessive traits: arginine: glycine amidinotransferase (AGAT) deficiency; OMIM 602360; and guanidinoacetate methyltransferase (GAMT) deficiency (OMIM 601240)) and one disorder of creatine transport (X-linked recessive SLC6A8 creatine transporter deficiency (OMIM 300036)). All these disorders are characterized by brain creatine deficiency, detectable by magnetic resonance spectroscopy. Affected patients can have mental retardation, hypotonia, autism or behavioral problems and seizures. The diagnosis of these conditions relies on the measurement of plasma and urine creatine and guanidinoacetate. Creatine levels in plasma are reduced in both creatine synthesis defects and guanidinoacetate is increased in GAMT deficiency. The urine creatine/creatinine ratio is elevated in creatine transporter deficiency with normal plasma levels of creatine and guanidinoacetate. The diagnosis is confirmed in all cases by DNA testing or functional studies. Defects of creatine biosynthesis are treated with creatine supplements and, in GAMT deficiency, with ornithine and dietary restriction of arginine through limitation of protein intake. No causal therapy is yet available for creatine transporter deficiency and supplementation with the guanidinoacetate precursors arginine and glycine is being explored. The excellent response to therapy of early identified patients with GAMT or AGAT deficiency candidates these condition for inclusion in newborn screening programs.

l-arginine:glycine amidinotransferase (AGAT) deficiency: clinical presentation and response to treatment in two patients with a novel mutation.

Creatine and creatine phosphate provide storage and transmission of phosphate-bound energy in muscle and brain. Of the three inborn errors of creatine metabolism causing brain creatine depletion, l-arginine:glycine amidinotransferase (AGAT) deficiency has been described in only two families. We describe clinical and biochemical features, magnetic resonance spectroscopy (MRS) findings and response to creatine supplementation in two siblings with a novel mutation in the AGAT-encoding GATM gene. The sister and brother were evaluated at age 12 and 18years, respectively, because of mild mental retardation, muscle weakness and low weight. Extensive work-up had previously yielded negative results. Electron microscopy of the muscle revealed tubular aggregates and the activity of respiratory chain complexes was decreased in the muscle. Urine organic acid concentrations normalized to urine creatinine concentration were all increased, suggesting a creatine metabolism disorder. Brain MRS was remarkable for absence of creatine. Urine guanidinoacetate levels by tandem mass spectrometry were low, suggesting AGAT deficiency. GATM sequencing revealed a homozygous single nucleotide insertion 1111_1112insA, producing a frame-shift at Met-371 and premature termination at codon 376. Eleven months after commencing treatment with oral creatine monohydrate 100mg/kg/day, repeat MRI/MRS showed significantly increased brain creatine in the sister and a slight increase in the older brother. The parents' impression of improved strength and stamina was substantiated by increased post-treatment versus pre-treatment scores in the Vineland Adaptive Behavior Scale, straight-arm raising and timed up-and-go tests. Similarly, there was an apparent improvement in cognitive function, with significantly increased IQ-scores in the sister and marginal improvement in the brother.

Arginine:glycine amidinotransferase (AGAT) deficiency in a newborn: early treatment can prevent phenotypic expression of the disease.

Arginine:glycine amidinotransferase deficiency is a treatable inborn error of creatine synthesis, characterized by mental retardation, language impairment, and behavioral disorders. We describe a patient in whom arginine:glycine amidinotransferase was diagnosed at birth and treated at 4 months with creatine supplementation. In contrast with his 2 older sisters, he had normal psychomotor development at 18 months.

Gatm, a creatine synthesis enzyme, is imprinted in mouse placenta.

To increase our understanding of imprinting and epigenetic gene regulation, we undertook a search for new imprinted genes. We identified Gatm, a gene that encodes l-arginine:glycine amidinotransferase, which catalyzes the rate-limiting step in the synthesis of creatine. In mouse, Gatm is expressed during development and is imprinted in the placenta and yolk sac, but not in embryonic tissues. The Gatm gene maps to mouse chromosome 2 in a region not previously shown to contain imprinted genes. To determine whether Gatm is located in a cluster of imprinted genes, we investigated the expression pattern of genes located near Gatm: Duox1-2, Slc28a2, Slc30a4 and a transcript corresponding to LOC214616. We found no evidence that any of these genes is imprinted in placenta. We show that a CpG island associated with Gatm is unmethylated, as is a large CpG island associated with a neighboring gene. This genomic screen for novel imprinted genes has elucidated a new connection between imprinting and creatine metabolism during embryonic development in mammals.

Creatine depletion in a new case with AGAT deficiency: clinical and genetic study in a large pedigree.

Arginine:glycine amidinotransferase (AGAT, EC 2.1.4.1) deficiency is a recently recognized autosomal recessive inborn error of creatine biosynthesis, characterized by mental retardation and severe language impairment. We extensively investigated a third 5-year-old patient with AGAT deficiency, discovered in the pedigree of the same Italian family as the two index cases. At the age of 2 years he presented with psychomotor and language delay, and autistic-like behavior. Brain MRI was normal, but brain 1H-MRS disclosed brain creatine depletion, which almost completely normalized following creatine monohydrate supplementation. A remarkable clinical improvement paralleled the restoration of brain creatine concentration. AGAT and GAMT (guanidinoacetate:methyltransferase) genes were analyzed in the proband and in 26 relatives, including the two cousins with AGAT deficiency. Sequencing of the proband's AGAT gene disclosed the same homozygous mutation at nt position 9093 converting a tryptophan (TGG) to a stop codon (TAG) at residue 149 (W149X), as already described in the two previously reported cases. The proband's parents and 10 additional subjects of the pedigree were carriers for this mutation. AGAT deficiency was further confirmed by undetectable AGAT activity in the patient's lymphoblasts. Mutation analysis of the GAMT gene revealed a sequence variation in exon 6 (T209M), not in the proband, but in 15 additional subjects from the pedigree. The silent nature of this sequence variation is supported by its homozygosity in one AGAT deficient cousin and in one asymptomatic adult, both with normal GAMT activity.

Arginine:glycine amidinotransferase deficiency: the third inborn error of creatine metabolism in humans.

Arginine:glycine amidinotransferase (AGAT) catalyzes the first step of creatine synthesis, resulting in the formation of guanidinoacetate, which is a substrate for creatine formation. In two female siblings with mental retardation who had brain creatine deficiency that was reversible by means of oral creatine supplementation and had low urinary guanidinoacetate concentrations, AGAT deficiency was identified as a new genetic defect in creatine metabolism. A homozygous G-A transition at nucleotide position 9297, converting a tryptophan codon (TGG) to a stop codon (TAG) at residue 149 (T149X), resulted in undetectable cDNA, as investigated by reverse-transcription PCR, as well as in undetectable AGAT activity, as investigated radiochemically in cultivated skin fibroblasts and in virus-transformed lymphoblasts of the patients. The parents were heterozygous for the mutant allele, with intermediate residual AGAT activities. Recognition and treatment with oral creatine supplements may prevent neurological sequelae in affected patients.

Expression of Xenopus L-arginine:glycine amidinotransferase (XAT) during early embryonic development.

We have isolated a full-length cDNA encoding Xenopus L-arginine:glycine amidinotransferase (XAT), which shares a highly conserved sequence with human, chick and rat amidinotransferase. Although there are some studies about its structure and function in energy metabolism of adult tissues in some other species, little is known about its roles during early embryonic development. Characterization of embryonic expression indicates that XAT is differentially expressed around the yolk plug including the dorsal blastopore area at early gastrula stages and is extensively expressed in the midline of the neural plate of early neurula stages. Sections reveal that its transcripts are located in the notochord. In the tailbud stage signals are found both in the notochord and trunk area, whereas only faint signals can be found in the cephalic part.

Cloning and sequencing of rat kidney L-arginine:glycine amidinotransferase. Studies on the mechanism of regulation by growth hormone and creatine.

L-Arginine-glycine amidinotransferase (transamidinase) is the first and rate-limiting step in creatine biosynthesis. Rats fed a creatine-supplemented diet or hypophysectomized rats have only 20% of the kidney transamidinase activity as intact rats fed a creatine-free diet. A cDNA clone corresponding to transamidinase was isolated by immunoscreening of a lambda gt11 expression library prepared from rat kidney mRNA. The transamidinase cDNA had an open reading frame containing the known sequence of the amino-terminal peptide of transamidinase. Based on the cDNA sequence, transamidinase is synthesized as a precursor with an amino-terminal extension of 50 amino acids, consistent with its mitochondrial localization. Comparison of the transamidinase sequence with the protein data base identified only a single, related protein. Remarkably, this protein, which has a 37% amino acid identity with transamidinase, is also an amidinotransferase, catalyzing streptomycin biosynthesis in Streptomyces griseus. Transamidinase cDNA was used to investigate the regulation of mRNA levels by creatine and growth hormone. Hypophysectomized rats were fed a creatine-free or a creatine-supplemented diet and maintained with and without injections of growth hormone. An excellent correlation was found between changes in transamidinase activity and mRNA levels in response to creatine and growth hormone. Thus, the regulation of transamidinase by creatine and growth hormone is at a pretranslational level. In addition, the two effectors do not act independently but interact at a pretranslational level to control transamidinase gene expression.

The amino acid sequences of human and pig L-arginine:glycine amidinotransferase.

We have isolated and sequenced the L-arginine:glycine amidinotransferase of pig kidney mitochondria. Due to endogenous proteolysis, the purified molecules showed some heterogeneity at the N terminus. The longest form recovered had 386 amino acids. Part of the pig amidinotransferase sequence information was used to isolate cDNA clones coding for the human enzyme. The deduced amino acid of the human amidinotransferase was 37 amino acids longer due to the presence of a single sequence. The mature proteins were 94% identical to each other and 36% identical to the sequences of bacterial L-arginine:inosamine phosphate amidinotransferases.