Creatine-supplemented Diet Research Articles

Effects of Creatine Supplementation on the Myostatin Pathway and Myosin Heavy Chain Isoforms in Different Skeletal Muscles of Resistance-Trained Rats

Creatine has been used to maximize resistance training effects on skeletal muscles, including muscle hypertrophy and fiber type changes. This study aimed to evaluate the impact of creatine supplementation on the myostatin pathway and myosin heavy chain (MyHC) isoforms in the slow- and fast-twitch muscles of resistance-trained rats. Twenty-eight male Wistar rats were divided into four groups: a sedentary control (Cc), sedentary creatine supplementation (Cr), resistance training (Tc), and resistance training combined with creatine supplementation (Tcr). Cc and Tc received standard commercial chow; Cr and Tcr received a 2% creatine-supplemented diet. Tc and Tcr performed a resistance training protocol on a ladder for 12 weeks. Morphology, MyHC isoforms, myostatin, follistatin, and ActRIIB protein expressions were analyzed in soleus and white gastrocnemius portion samples. The results were analyzed using two-way ANOVA and Tukey’s test. Tc and Tcr exhibited higher performance than their control counterparts. Resistance training increased the ratio between muscle and body weight, the cross-sectional area, as well as the interstitial collagen fraction. Resistance training alone increased MyHC IIx and follistatin while reducing myostatin (p < 0.001) and ActRIIB (p = 0.040) expressions in the gastrocnemius. Resistance training induced skeletal muscle hypertrophy and interstitial remodeling, which are more evident in the gastrocnemius muscle. The effects were not impacted by creatine supplementation.

Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure.

Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. Study 1: homoarginine deficiency-female AGAT-/- and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT-/- had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6-7weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency-as before, but AGAT-/- mice were given creatine-supplemented diet until 1week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial.

Role of creatine supplementation on the myofiber characteristics and muscle protein synthesis of grass carp (Ctenopharyngodon idellus).

To assess the role of dietary creatine on myofiber characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88.47 ± 1.44 g) creatine-supplemented diets (1.84, 5.91, 8.48, and 15.44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8.48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum, and improved hardness and chewiness of muscle due to shorter myofiber mean diameter, higher myofiber density and the frequencies of the diameters of class I and III and collagen content, longer sarcomere length, and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8.48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AAs), essential AAs, and free flavor AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen, and peroxisome proliferator-activated receptor-γ coactlvator-1α in muscle, and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8.48 mg/kg creatine improved fish health and skeletal muscle growth, and increased hardness and protein synthesis in muscle of grass carp by affecting myofiber characteristics and the TOR signaling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8.48 and 12.04 g/kg.

Dietary creatine requirement of red drum (Sciaenops ocellatus) and effects of water salinity on responses to creatine supplementation

Creatine is a multifunctional organic acid necessary for various physiological processes and is commonly supplemented to improve muscle growth and performance of humans. However, the effects of supplemented creatine in the diets of fishes is unclear. Thus, two feeding trials were conducted to examine the performance of juvenile red drum fed diets supplemented with creatine. The first feeding trial was conducted for 7 weeks, to determine the optimal dietary supplementation of creatine for red drum at low (5 ppt) salinity. Red drum is a euryhaline species and potential benefits of creatine may include enhanced energy utilization during osmoregulation. Therefore, the second trial was a two-part, 12-week, feeding trial. During the first 7 weeks, salinity was maintained at 3 ppt (low salinity), then, for an additional 5 weeks, maintained at 15 ppt (moderate salinity). The basal diet for both feeding trials was formulated to contain 40% crude protein, contributed equally by menhaden fishmeal and soy protein concentrate, and 10% lipid. For trial 1, five experimental diets were derived from the basal diet by supplementing creatine at 0.5, 1, 1.5, 2, or 4% by weight at the expense of cellulose. The diets for trial 1 were adjusted with a 50/50 blend of aspartate and glutamate to maintain the diets isonitrogenous. For trial 2, the same basal diet and one supplemented with creatine at 2% were prepared with the creatine-supplemented diet adjusted with a 50/50 blend of aspartate and glutamate on an equal-weight basis. In trial 1, significant (P ≤ .05) positive trends were observed in weight gain, feed efficiency, and survivability of fish fed the diets supplemented with creatine. Broken-line regression analysis indicated creatine supplementation at 1.98% of diet provided optimal weight gain of red drum. In trial 2, there was a trending increase in percent weight gain and feed efficiency of fish fed the creatine supplemented diet during the moderate salinity portion of the trial, resulting in significant improvements over the 12-week period. Results from these study confirm that red drum benefit from dietary creatine supplementation in practical diets.

Maternal creatine supplementation during pregnancy prevents acute and long-term deficits in skeletal muscle after birth asphyxia: a study of structure and function of hind limb muscle in the spiny mouse.

Maternal antenatal creatine supplementation protects the brain, kidney, and diaphragm against the effects of birth asphyxia in the spiny mouse. In this study, we examined creatine's potential to prevent damage to axial skeletal muscles. Pregnant spiny mice were fed a control or creatine-supplemented diet from mid-pregnancy, and 1 d before term (39 d), fetuses were delivered by c-section with or without 7.5 min of birth asphyxia. At 24 h or 33 ± 2 d after birth, gastrocnemius muscles were obtained for ex-vivo study of twitch-tension, muscle fatigue, and structural and histochemical analysis. Birth asphyxia significantly reduced cross-sectional area of all muscle fiber types (P < 0.05), and increased fatigue caused by repeated tetanic contractions at 24 h of age (P < 0.05). There were fewer (P < 0.05) Type I and IIa fibers and more (P < 0.05) Type IIb fibers in male gastrocnemius at 33 d of age. Muscle oxidative capacity was reduced (P < 0.05) in males at 24 h and 33 d and in females at 24 h only. Maternal creatine treatment prevented all asphyxia-induced changes in the gastrocnemius, improved motor performance. This study demonstrates that creatine loading before birth protects the muscle from asphyxia-induced damage at birth.

Maternal Creatine Supplementation during Pregnancy Prevents Long-Term Changes in Diaphragm Muscle Structure and Function after Birth Asphyxia.

Using a model of birth asphyxia, we previously reported significant structural and functional deficits in the diaphragm muscle in spiny mice, deficits that are prevented by supplementing the maternal diet with 5% creatine from mid-pregnancy. The long-term effects of this exposure are unknown. Pregnant spiny mice were fed control or 5% creatine-supplemented diet for the second half of pregnancy, and fetuses were delivered by caesarean section with or without 7.5 min of in-utero asphyxia. Surviving pups were raised by a cross-foster dam until 33±2 days of age when they were euthanized to obtain the diaphragm muscle for ex-vivo study of twitch tension and muscle fatigue, and for structural and enzymatic analyses. Functional analysis of the diaphragm revealed no differences in single twitch contractile parameters between any groups. However, muscle fatigue, induced by stimulation of diaphragm strips with a train of pulses (330ms train/sec, 40Hz) for 300sec, was significantly greater for asphyxia pups compared with controls (p<0.05), and this did not occur in diaphragms of creatine + asphyxia pups. Birth asphyxia resulted in a significant increase in the proportion of glycolytic, fast-twitch fibres and a reduction in oxidative capacity of Type I and IIb fibres in male offspring, as well as reduced cross-sectional area of all muscle fibre types (Type I, IIa, IIb/d) in both males and females at 33 days of age. None of these changes were observed in creatine + asphyxia animals. Thus, the changes in diaphragm fatigue and structure induced by birth asphyxia persist long-term but are prevented by maternal creatine supplementation.

Maternal creatine supplementation from mid-pregnancy protects the newborn spiny mouse diaphragm from intrapartum hypoxia-induced damage

We hypothesized that maternal creatine supplementation from mid-pregnancy would protect the diaphragm of the newborn spiny mouse from the effects of intrapartum hypoxia. Pregnant mice were fed a control or 5% creatine-supplemented diet from mid-gestation. On the day before term, intrapartum hypoxia was induced by isolating the pregnant uterus in a saline bath for 7.5-8 min before releasing and resuscitating the fetuses. Surviving pups were placed with a cross-foster dam, and diaphragm tissue was collected at 24 h postnatal age. Hypoxia caused a significant decrease in the cross-sectional area (∼19%) and contractile function (26.6% decrease in maximum Ca2=-activated force) of diaphragm fibers. The mRNA levels of the muscle mass-regulating genes MuRF1 and myostatin were significantly increased (2-fold). Maternal creatine significantly attenuated hypoxia-induced fiber atrophy, contractile dysfunction, and changes in mRNA levels. This study demonstrates that creatine loading before birth significantly protects the diaphragm from hypoxia-induced damage at birth.

Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo.

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

Abstract 3565: Regulation of Myocardial Creatine Uptake by Intra- and Extracellular Creatine Levels

Creatine plays an important role in energy metabolism in the heart. Cardiomyocytes accumulate creatine via a specific creatine transporter (CrT), the capacity of which is reduced in the failing heart, resulting in lower myocardial creatine concentration. To gain insight into how the CrT is regulated by intra- and extracellular creatine concentrations, we studied two mouse models of severely altered myocardial creatine levels, guanidinoacetate-N-methyl transferase knock out (GAMT−/−) mice and mice overexpressing the myocardial CrT (CrT-OE). We measured cardiac creatine uptake level in isolated creatine free GAMT−/− mouse hearts from using 14C-radiolabeled creatine, and found that creatine uptake capacity was massively elevated, with a 7-fold increase in Vmax. Cardiac creatine uptake levels in CrT-OE mice were increased on average 2.7-fold, showing a considerable variation, in line with a similar variation in creatine content as measured with HPLC. To understand whether the elevated CrT uptake was due to transcriptional regulation, we measured CrT mRNA levels in the GAMT−/− mouse hearts, but found only a 1.4-fold increase relative to WT values, and no significant difference relative to CrT mRNA levels in the CrT-OE mice. However, the increase in Cr uptake and in CrT mRNA levels in the GAMT−/− mice was prevented when mice were fed a creatine-supplemented diet, indicating that creatine uptake is subject to negative feedback regulation. To determine whether high intracellular creatine concentrations could downregulate CrT transcription, we correlated the expression of total and endogenous CrT mRNA with myocardial creatine levels in the CrT-OE mouse hearts. Total CrT mRNA (transgenic CrT + endogenous CrT) levels correlated well with myocardial creatine content (r=0.67; p&lt;0.0001), but endogenous CrT mRNA levels did not correlate at all with myocardial creatine content (r=0.01;p=0.96), suggesting that intracellular creatine levels did not influence CrT transcription. In summary, creatine uptake can be massively upregulated in the heart and this upregulation is subject to feedback inhibition. In addition, our results strongly suggest that CrT activity is predominantly regulated by mechanisms other than alterations in gene expression.

Oral creatine supplementation attenuates L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rats

L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson patients after a prolonged treatment with levodopa (L-DOPA). Since previous transcriptome and proteomic studies performed in the rat model of LID suggested important changes in striatal energy-related components, we hypothesize that oral creatine supplementation could prevent or attenuate the occurrence of LID. In this study, 6-hydroxydopamine-lesioned rats received a 2% creatine-supplemented diet for 1 month prior to L-DOPA therapy. During the 21 days of L-DOPA treatment, significant reductions in abnormal involuntary movements (AIMs) have been observed in the creatine-supplemented group, without any worsening of parkinsonism. In situ hybridization histochemistry and immunohistochemistry analysis of the striatum also showed a reduction in the levels of prodynorphin mRNA and FosB/ΔFosB-immunopositive cells in creatine-supplemented diet group, an effect that was dependant on the development of AIMs. Further investigation of the bioenergetics’ status of the denervated striatum revealed significant changes in the levels of creatine both after L-DOPA alone and with the supplemented diet. In conclusion, we demonstrated that combining L-DOPA therapy with a diet enriched in creatine could attenuate LID, which may represent a new way to control the motor complications associated with L-DOPA therapy.

Maternal creatine: does it reach the fetus and improve survival after an acute hypoxic episode in the spiny mouse (Acomys cahirinus)?

We hypothesized that elevating creatine in the maternal diet would reach fetal and placental tissues and improve fetal survival after acute hypoxia at birth. Pregnant spiny mice were fed a control or 5% creatine-supplemented diet from day 20 of gestation (term, approximately 39 days). On days 37-38, intrauterine hypoxia was induced by placement of the isolated uterus in a saline solution bath for 7.5-8 minutes, after which fetuses were expelled from the uterus and resuscitation was attempted by manual palpation of the chest. Total creatine content (creatine + phosphocreatine) of placental, fetal, and maternal organs was measured. The maternal creatine diet significantly increased total creatine content in the placenta, fetal brain, heart, liver, and kidney and increased the capacity of offspring to survive birth hypoxia. Maternal creatine improved postnatal growth after birth hypoxia. This study provides evidence that creatine has potential as a prophylactic therapy for pregnancies that are classified as high risk for fetal hypoxia.

Creatine supplementation decreases homocysteine in an animal model of uremia

Hyperhomocysteinemia is prevalent in more than 85% of patients with end-stage renal disease (ESRD) and is thought to contribute to the excess cardiovascular mortality and morbidity. Creatine is synthesized by methylation of guanidinoacetate with formation of S-adenosylhomocysteine and subsequently, homocysteine (Hcy). Creatine supplementation down-regulates its endogenous synthesis and, thus, may reduce Hcy production. The present study investigates the effect of creatine supplementation on Hcy concentrations in an animal model of uremia. Male Wistar rats were either sham-operated and received a control diet (N = 8) or a 2% creatine-supplemented diet (N = 8), or underwent subtotal nephrectomy and received a control diet (N = 10) or a 2%-supplemented creatine diet (N = 10). After 2 weeks of treatment, total plasma Hcy, creatine, creatinine, folate, and vitamin B12 were determined, as well as hepatic folate and vitamin B12 concentrations. Plasma creatinine concentrations were higher in nephrectomized animals, but similar in creatine-supplemented and control diet-fed animals. Plasma Hcy was higher in nephrectomized animals but lower in creatine-supplemented nephrectomized animals compared to nephrectomized control diet-fed animals (12.1 +/- 2.4 micromol/L vs. 15.4 +/- 1.7 micromol/L; P < 0.01). Total plasma Hcy inversely correlated with plasma creatine concentrations (r =-0.39; P = 0.02). Plasma folate was higher in supplemented animals and hepatic tetrahydrofolate (THF) was higher in nephrectomized supplemented animals. Plasma vitamin B12 was similar in all groups, whereas hepatic vitamin B12 was higher in nephrectomized animals. Creatine supplementation can effectively lower plasma Hcy concentrations in an animal model of uremia and should be further investigated as a potential treatment for hyperhomocysteinemia in patients with ESRD.

Creatine supplementation does not affect kidney function in an animal model with pre-existing renal failure

Creatine is widely used as an ergogenic substance among athletes. Safety of prolonged creatine intake has been questioned, based upon case reports and animal data. We investigated the effect of prolonged creatine ingestion on renal function in animals with normal kidney function or pre-existing kidney failure, respectively. Male Wistar rats were randomly allocated to four experimental groups: (i) sham-operated, control diet; (ii) sham-operated, creatine-supplemented diet (2% w/w (0.9+/-0.2 g creatine/kg body weight/day)); (iii) two-thirds nephrectomized, control diet; and (iv) two-thirds nephrectomized, creatine supplemented diet. Glomerular filtration rate was determined using inulin and creatinine clearance, together with albumin excretion, urea clearance, muscle and serum creatine and serum cystatin C concentrations. In contrast to previous reports, no detrimental effects of creatine supplementation on the renal function indices were observed in two-thirds nephrectomized or sham-operated animals. No differences were observed in inulin (0.28+/-0.08 vs 0.25+/-0.08 ml/min/100 g; P=NS) or creatinine clearance rates. Serum cystatin C concentration, urinary protein excretion, and albumin and urea clearance were comparable between creatine-supplemented and control-diet fed animals in both sham-operated and two-thirds nephrectomized animals. Serum creatine and intramuscular total creatine concentrations were higher in creatine-supplemented groups (P<0.05). Creatine supplementation at a dosage of 2% w/w for 4 weeks does not impair kidney function in animals with pre-existing renal failure or in control animals.

Creatine-supplemented diet extends Purkinje cell survival in spinocerebellar ataxia type 1 transgenic mice but does not prevent the ataxic phenotype.

It is not known why expression of a protein with an expanded polyglutamine region is pathogenic in spinocerebellar ataxia, Huntington's disease and several other neurodegenerative diseases. Dietary supplementation with creatine improves survival and motor performance and delays neuronal atrophy in the R6/2 transgenic mouse model of Huntington's disease. These effects may be due to improved energy and calcium homeostasis, enhanced presynaptic glutamate uptake, or protection of mitochondria from the mitochondrial permeability transition. We tested the effects of a 2% creatine-supplemented diet and treatment with taurine-conjugated ursodeoxycholic acid, a bile constituent that can inhibit the mitochondrial permeability transition, on ataxia and Purkinje cell survival in a transgenic model of spinocerebellar ataxia type 1. After 24 weeks, transgenic mice on the 2% creatine diet had cerebellar phosphocreatine levels that were 72.5% of wildtype controls, compared to 26.8% in transgenic mice fed a control diet. The creatine diet resulted in maintenance of Purkinje cell numbers in these transgenic mice at levels comparable to wildtype controls, while transgenic mice fed a control diet lost over 25% of their Purkinje cell population. Nevertheless, the ataxic phenotype was neither improved nor delayed. Repeated s.c. ursodeoxycholic acid injections markedly elevated ursodeoxycholic acid levels in the brain without adverse effects, but provided no improvement in phenotype or cell survival in spinocerebellar ataxia type 1 mice. These results demonstrate that preserving neurons from degeneration is insufficient to prevent a behavioral phenotype in this transgenic model of polyglutamine disease. In addition, we suggest that the means by which creatine mitigates against the neurodegenerative effects of an ataxin-1 protein containing an expanded polyglutamine region is through mechanisms other than stabilization of mitochondrial membranes.

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.