Creatine and the creatine transporter: a review

The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the expression of the CreaT1 protein in several tissues including neural, cardiac and skeletal muscle. There is very little information available about the factors regulating CreaT gene expression. There is some evidence that suggests the intracellular Cr concentration may be involved in the regulatory process but there is much more to learn before this process is understood. The activity of the CreaT protein is controlled by many factors. These include substrate concentration, transmembrane Na+ gradients, cellular location, and various hormones. It is also likely that transporter activity is influenced by its phosphorylation state and by its interaction with other plasma membrane proteins. The extent of CreaT protein glycosylation may vary within cells, the functional significance of which remains unclear.

Creatine transporter protein content, localization, and gene expression in rat skeletal muscle

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P <= 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P <= 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P <= 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P <= 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.

Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells

Zinc is an essential trace element required by all living organisms. An adequate supply of zinc is particularly important in the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. The mechanisms by which zinc becomes a constituent of breast milk have not been elucidated. The function of the zinc transporter ZnT4 in the transport of zinc into milk during lactation was previously demonstrated by studies of a mouse mutant, the ‘lethal milk’ mouse, where a mutation in the ZnT4 gene decreased the transport of zinc into milk. In the present study, we have investigated the expression of the human orthologue of ZnT4 (hZnT4) in the human breast. We detected hZnT4 mRNA expression in the tissue from the resting and lactating human breast, using reverse-transcriptase PCR. Western-blot analysis using antibodies to peptide sequences of hZnT4 detected a major band of the predicted size of 47 kDa and a minor band of 77 kDa, in extracts from the resting and lactating breast tissues. There was no difference in the hZnT4 expression levels between lactating and resting breasts. The hZnT4 protein was present in the luminal cells of the ducts and alveoli where it had a granular distribution. A cultured human breast epithelial cell line PMC42 was used to investigate the subcellular distribution of hZnT4 and this showed a granular label throughout the cytoplasm, consistent with a vesicular localization. The presence of zinc-containing intracellular vesicles was demonstrated by using the zinc-specific fluorphore Zinquin (ethyl-[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate). Double labelling indicated that there was no obvious overlap between Zinquin and the hZnT4 protein, suggesting that hZnT4 was not directly involved in the transport of zinc into vesicles. We detected expression of two other members of the hZnT family, hZnT1 and hZnT3, in human breast epithelial cells. We conclude that hZnT4 is constitutively expressed in the human breast and may be one of the several members of the ZnT family involved in the transport of zinc into milk.

Analysis of zinc transporter, hZnT4(Slc30A4), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.

Human skeletal muscle creatine transporter mRNA and protein expression in healthy, young males and females

The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 ± 5.0 years) and female subjects (n = 12, age: 21.7 ± 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (~ 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to ß-actin mRNA and the TCr content (males: 117.8 ± 2.2, females: 125.3 ± 4.3 mmol.kg^–1 dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.

Skeletal muscle total creatine content and creatine transporter gene expression in vegetarians prior to and following creatine supplementation

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.

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells

The cellular effects of biodiesel emissions particulate matter (BDEP) and petroleum diesel emissions particulate matter (PDEP) were compared using a human airway cell line, A549. At concentrations of 25 µg/ml, diesel particulate matter induced the formation of multinucleate cells. In cells treated with a mixture of 80% PDEP:20% BDEP, 52% of cells were multinucleate cells compared with only 16% of cells treated with 20% PDEP:80% BDEP with a background multinucleate rate of 7%. These results demonstrate a causal relation between the formation of multinucleate cells and exposure to exhaust particulate matter, in particular diesel exhaust. Exposure of A549 cells to PDEP induced apoptosis, seen by active caspase-3 expression and the presence of cleaved pancytokeratin. PDEP exhaust was a much stronger inducer of cellular death through apoptosis than BDEP. There was an eightfold increase in the expression of SLC30A3 (zinc transporter-3 or ZnT3) in cells exposed to 80% PDEP:20% BDEP compared to untreated cells. The increase in ZnT3 expression seen in apoptotic cells following PDEP suggests a role for this zinc transporter in the apoptotic pathway, possibly through controlling zinc fluxes. As exposure to diesel exhaust particles is associated with asthma and apoptosis in airway cells, diesel exhaust particles may directly contribute to asthma by inducing epithelial cell death through apoptotic pathway.

Developmental changes in the expression of creatine synthesizing enzymes and creatine transporter in a precocial rodent, the spiny mouse

Background : Creatine synthesis takes place predominately in the kidney and liver via a two-step process involving AGAT (L-arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase). Creatine is taken into cells via the creatine transporter (CrT), where it plays an essential role in energy homeostasis, particularly for tissues with high and fluctuating energy demands. Very little is known of the fetal requirement for creatine and how this may change with advancing pregnancy and into the early neonatal period. Using the spiny mouse as a model of human perinatal development, the purpose of the present study was to comprehensively examine the development of the creatine synthesis and transport systems.

Results : The estimated amount of total creatine in the placenta and brain significantly increased in the second half of pregnancy, coinciding with a significant increase in expression of CrT mRNA. In the fetal brain, mRNA expression of AGAT increased steadily across the second half of pregnancy, although GAMT mRNA expression was relatively low until 34 days gestation (term is 38–39 days). In the fetal kidney and liver, AGAT and GAMT mRNA and protein expression were also relatively low until 34–37 days gestation. Between mid-gestation and term, neither AGAT or GAMT mRNA or protein could be detected in the placenta.

Conclusion : Our results suggest that in the spiny mouse, a species where, like the human, considerable organogenesis occurs before birth, there appears to be a limited capacity for endogenous creatine synthesis until approximately 0.9 of pregnancy. This implies that a maternal source of creatine, transferred across the placenta, may be essential until the creatine synthesis and transport system matures in preparation for birth. If these results also apply to the human, premature birth may increase the risk of creatine deficiency.

Multiple urea transporter proteins in the kidney of holocephalan elephant fish (Callorhinchus milii)

Reabsorption of filtered urea by the kidney is essential for retaining high levels of urea in marine cartilaginous fish. Our previous studies on the shark facilitative urea transporter (UT) suggest that additional UT(s) comprising the urea reabsorption system could exist in the cartilaginous fish kidney. Here, we isolated three cDNAs encoding UTs from the kidney of elephant fish, Callorhinchus milii, and termed them efUT-1, efUT-2 and efUT-3. efUT-1 is orthologous to known elasmobranch UTs, while efUT-2 and efUT-3 are novel UTs in cartilaginous fish. Two variants were found for efUT-1 and efUT-2, in which the NH₂-terminal intracellular domain was distinct between the variants. Differences in potential phosphorylation sites were found in the variant-specific NH2-terminal domains. When expressed in Xenopus oocytes, all five UT transcripts including the efUT-1 and efUT-2 variants induced more than a 10-fold increase in [¹⁴C] urea uptake. Phloretin inhibited dose-dependently the increase of urea uptake, suggesting that the identified UTs are facilitative UTs. Molecular phylogenetic analysis revealed that efUT-1 and efUT-2 had diverged in the cartilaginous fish lineage, while efUT-3 is distinct from efUT-1 and efUT-2. The present finding of multiple UTs in elephant fish provides a key to understanding the molecular mechanisms of urea reabsorption system in the cartilaginous fish kidney.

Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a uglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy. 2-deoxy-[1-14C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

Exercise and skeletal muscle glucose transporter 4 expression: molecular mechanisms

1.      Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT-4) expression in human skeletal muscle.

2.      Glucose transporter isoform 4 gene expression is increased immediately following a single bout of exercise, and the GLUT-4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. Glucose transporter isoform enhancer factor and MEF2 DNA binding activities are increased following exercise, and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined.

3.      These studies find possible roles for histone deacetylase 5 (HDAC5), adenosine monophosphate–activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and p38 mitogen-activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation.

4.      Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterized by mitochondrial dysfunction and insulin resistance in skeletal muscle.

Creatine transporter expression in skeletal muscle

Creatine is an important molecule involved in providing energy to the body. Its major stores are in skeletal muscle. The creatine transporter protein (CreaT) mRNA is believed to be responsible for the uptake of the majority of creatine in skeletal muscle. This thesis examined factors that might have affected the expression of the creatine transporter in skeletal muscle.

The function of zinc transporter genes in human cells

Zinc is an essential nutrient for all living organisms and a deficiency may result in major health problems. This project provides insights into how zinc is processed by cells within the human body. The experiments also contribute to a better understanding of the molecular basis of zinc deficiency disorders. Novel procedures developed in this project may prove invaluable to the wider scientific community.