Creatine Analogue Beta-guanidinopropionic Acid Research Articles

Impairment of PGC-1alpha expression, neuropathology and hepatic steatosis in a transgenic mouse model of Huntington's disease following chronic energy deprivation

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1alpha function will exert therapeutic benefits in HD patients.

The Orally Active Antihyperglycemic Drug β-Guanidinopropionic Acid Is Transported by the Human Proton-Coupled Amino Acid Transporter hPAT1

The orally administered creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases plasma glucose levels by increasing the sensitivity to insulin. This effect is based on a beta-GPA induced expression of mRNA and total protein content of the insulin-responsive glucose transporter GLUT4. Although the oral availability of beta-GPA is well established, the underlying uptake mechanism has not yet been studied. We investigated whether the H(+)-coupled amino acid transporter PAT1, which is expressed in the apical membrane of intestinal cells, accepts guanidine derivatives as substrates. Uptake of l-[(3)H]proline into Caco-2 cells expressing hPAT1 constitutively was strongly inhibited by beta-GPA and its derivatives guanidinoacetic acid (GAA) and 4-guanidinobutyric acid (4-GBA). Competition assays revealed apparent affinity constants of about 1.5 mM. Electrophysiological measurements at hPAT1-expressing Xenopus laevis oocytes unequivocally demonstrated that beta-GPA, GAA and 4-GBA are effectively transported by this transport system in an electrogenic manner. We conclude that hPAT1 might be responsible for the intestinal absorption of beta-GPA thereby allowing its oral administration. Moreover, with beta-GPA we identified a new high affinity hPAT1 substrate that might be an interesting starting point for future drug design-drug delivery strategies.

In Vivo Effects of Myocardial Creatine Depletion on Left Ventricular Function Morphology and Lipid Metabolism: Study in a Mouse Model

Background The failing heart is characterized by disturbed myocardial energy metabolism and creatine depletion. The aims of this study were to evaluate in vivo the effects of creatine (Cr) depletion on 1) left ventricular (LV) function, morphology, and lipid metabolism and 2) to test whether functional, morphologic, and metabolic disturbances induced by Cr depletion are reversible. Methods and Results Male Balb/c mice ∼20 g were used. Two groups were studied: the mice treated with creatine analogue beta-guanidinopropionic acid (BGP) (n = 30) and controls (n = 30). BGP (1 M) were administered by subcutaneously implanted osmotic minipumps for 4 weeks. The mice were examined in vivo using echocardiography. High-performance liquid chromatography was used for measurements of the myocardial creatine, adenosine nucleotides, and lipids. BGP was discontinued in a subgroup of mice and these animals were followed for an additional 4 weeks, after which echocardiography was performed under resting and stress conditions. Body weight was lower in BGP mice ( P < .001) compared with the controls after 4 weeks. The total myocardial Cr pool was ∼40% lower ( P < .001), whereas total nucleotide pool (TAN) was 18% lower ( P = n.s.) in the BGP group. LV systolic function was disturbed at rest and stress in the BGP mice (both P < .05). LV dimensions and LV mass were increased in the BGP group ( P < .05). There was an accumulation of intracellular triglycerides in the BGP-treated mice ( P < .05). Four weeks after BGP discontinuation Cr, TAN and TG content were restored to the normal levels while LV function, dimension, and mass were normalized. Conclusions Myocardial Cr depletion results in LV dysfunction, pathologic remodeling, and lipid accumulation. These alterations are completely reversible on normalization of Cr content. Cr metabolism may be an important target for pharmacologic intervention to increase myocardial efficiency and structural integrity of the failing heart.

Skeletal Muscle Phosphocreatine Depletion Depresses Myocellular Energy Status During Sepsis

To determine the effects of phosphocreatine (PCr) depletion on myocellular energetics. Randomized controlled study. University laboratory. Thirty-eight adult male Wistar rats (110-121 g). The poorly metabolized creatine analogue beta-guanidinopropionic acid, (beta-GPA, 2% of a gel diet) was fed to the rats for 14 days to replace (75%) endogenous PCr stores before cecal ligation and puncture (CLP). Rats were randomized to receive sham operation and gel diet (sham-gel group [n=10]), sham operation and beta-GPA diet (sham-beta-GPA group [n=9]), CLP and gel diet (CLP-gel group [n=10]), and CLP and beta-GPA diet (CLP-beta-GPA group [n=9]). On day 14, all animals underwent operation. Twenty-four hours later, in vivo phosphorus 31-labeled magnetic resonance spectroscopy (31P-MRS) of the gastrocnemius muscle was performed. Muscle samples were collected to determine enzyme activities of beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, citrate synthase, and the metabolites adenosine triphosphate (ATP), PCr, inorganic phosphate, and creatine. Free adenosine diphosphate levels, the phosphorylation potential, and free energy change of ATP hydrolysis were then calculated. All animals undergoing CLP but no controls had positive results of blood cultures. Although sham-beta-GPA animals had altered bioenergetics, CLP-beta-GPA rats experienced a greater deterioration of energy state compared with CLP-gel controls. Glycolytic and oxidative enzyme activities were not significantly different between groups and therefore could not explain the observed differences. There is an overall decrease in energy availability during sepsis, which is worsened by PCr depletion. These changes support the contention that PCr plays an important role as an ATP buffer during systemic infection.

Creatine analogue beta-guanidinopropionic acid alters skeletal muscle AMP deaminase activity.

Dietary supplementation of the creatine analogue beta-guanidinopropionic acid (beta-GPA) decreases in vitro skeletal muscle AMP deaminase (AMP-D) activity in rats. Downregulation of AMP-D activity was progressive and greater in fast-twitch muscles (70-80%) than in the slow-twitch soleus muscle (approximately 50%). The loss in AMP-D activity had little effect on inosine 5'-monophosphate accumulation in mixed-fiber muscle with intense tetanic contractions. In contrast, inosine 5'-monophosphate formation was evident earlier in fast-twitch red and white fiber sections of creatine-depleted animals during intense twitch contractions, indicating that fast-twitch muscle of beta-GPA-treated rats buffers decreases in the ATP/ADPfree ratio via deamination, even though AMP-D activity is less. Isoforms of skeletal muscle AMP-D mRNAs in mixed-fiber muscle were not altered by feeding beta-GPA for up to 9 wk. Creatine depletion did not alter total immunoreactivity; however, a redistribution of AMP-D immunoreactivity from primarily an approximately 80-kDa form toward lower apparent molecular mass species (approximately 60 and approximately 56 kDa) was observed. Posttranslational changes in AMP-D appear related to changes in activity.

Metabolite utilization and compartmentation in porcine carotid artery: a study using beta-guanidinopropionic acid

The relationship between substrate and metabolism in vascular smooth muscle has been investigated by studying the acute energetic effects caused by the creatine analogue beta-guanidinopropionic acid (beta-GPA) on porcine carotid arteries using 31P-nuclear magnetic resonance (NMR). Porcine carotid arteries were superfused for 12 h with Krebs-Henseleit buffer at 22 degrees C, containing 50 mM beta-GPA, and either 11 mM glucose or 5 mM pyruvate as substrate. beta-GPA enters the cells and becomes phosphorylated by creatine kinase to produce beta-GPA-P. Perfusion with beta-GPA leads to the formation of NMR observable beta-GPA-P (after 2.5 h). The appearance of beta-GPA-P with time was significantly greater when glucose was used as substrate. To differentiate between oxidative and glycolytic metabolism in the phosphorylation of beta-GPA, 1 mM cyanide was included in the perfusion buffer containing 50 mM beta-GPA and 11 mM glucose. No phosphocreatine (PCr) was observed with these conditions, and there was a small but significant decrease in ATP concentration ([ATP]) compared with glucose perfusion without cyanide (0.56 +/- 0.02 to 0.47 +/- 0.02 mumol/g wet wt), that was greater than the concentration with pyruvate as substrate (0.25 +/- 0.03 mumol/g wet wt). Thus the [ATP] during cyanide treatment is maintained with glycolytic metabolism. Despite the relatively high [ATP], accumulation of beta-GPA-P only occurred over a much slower time course ( > 10 h) than without cyanide.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of contractile properties of extensor digitorum longus in response to creatine-analogue administration and/or hindlimb suspension in rats.

Changes of contractile properties of extensor digitorum longus in response to hindlimb suspension and/or altered high-energy phosphate contents were studied in rats. A reduction of high-energy phosphates, especially phosphocreatine, was seen in rats fed creatine analogue beta-guanidinopropionic acid (beta-GPA), but they were elevated after 10-d supplementation of creatine. The one-half relaxation time was increased by feeding beta-GPA, but was normalized by creatine supply. The fatigue resistance of creatine-depleted muscle was significantly improved, but tended to decrease by suspension and creatine supply, although it was still better than that in the control diet group. It is indicated that the contractile properties of muscle are influenced by the high-energy phosphate content. It is also suggested that the endurance capacity may be influenced by the mitochondrial respiratory capacity, but not necessarily by the levels of high-energy phosphates.

Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle.

This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid [creatine depletion juvenile (CDJ) group]. Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens [normal diet adult and creatine depletion adult (CDA) groups]. After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC distribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC transitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of animals to be used.

Interaction of chronic creatine depletion and muscle unloading: effects on postural locomotor muscles

In some rodent skeletal muscles, hindlimb non-weight-bearing activity induces a shift in the expression of myosin heavy chains (MHCs) that favors the type II isoforms at the expense of type I. Chemically induced chronic creatine depletion results in isomyosin shifts favoring expression of type I MHCs. In this study, creatine depletion was induced separately and in combination with non-weight-bearing activity to determine if the response to lowering this metabolite would counter the MHC transitions expected from non-weight bearing. Creatine depletion was induced by feeding rats a diet supplemented with the creatine analogue beta-guanidinopropionic acid (beta-GPA). Female Sprague-Dawley rats weighing 247 +/- 8 g were randomly assigned to four groups: 1) normal diet control, 2) beta-GPA control (BC), 3) normal diet suspended (NS), and 4) beta-GPA suspended (BS). BC and BS animals were fed a diet containing the creatine analogue for 68 days. Hindlimb non-weight bearing in BS and NS animals was accomplished by tail suspension for the final 30 days of this period. beta-GPA feeding lowered the creatine content of muscles sampled by 65%. Creatine depletion resulted in a 16% increase in citrate synthase activity in the soleus (SOL) and a 24% increase in the plantaris (PLN). In two postural muscles, the SOL and vastus intermedius (VI), tail suspension resulted in large decreases in the type I MHC expression and increases in type IIx and IIb MHCs. In two locomotor muscles, the PLN and medial gastrocnemius, type I MHC declined and type IIb increased with suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile economy and aerobic recovery metabolism in skeletal muscle adapted to creatine depletion

Mice were treated for 7-12 wk with the creatine analogue beta-guanidinopropionic acid (beta-GPA). Treatment reduced total creatine to approximately 5% of control values in soleus (SOL) and extensor digitorum longus (EDL) muscles. In both muscles from treated mice, phosphorylated beta-GPA accumulated and resting [ATP] decreased by approximately 50%. Relative to controls, cytochrome oxidase and citrate synthase activities increased significantly in EDL from treated mice, but not in SOL; creatine kinase activity decreased significantly in SOL, but not in EDL. Measurements of poststimulation energy metabolism show that the energy cost to maintain tension in SOL and EDL from treated mice was approximately 50% of that in control muscle. Relative to controls, first-order rate constants of poststimulation O2 demand were 2- and 3.6-fold greater in SOL and EDL, respectively, from treated mice. Increased economy of SOL and EDL from treated mice is consistent with previously reported changes in myosin isoenzymes. Increases in rate constants of O2 utilization in creatine-depleted muscle are inconsistent with the hypothesis that cytoplasmic or mitochondrial creatine kinase is rate limiting for cellular respiration.

Actions of the creatine analogue β-guanidinopropionic acid on rat heart mitochondria

The action of the creatine analogue beta-guanidinopropionic acid (beta-GPA) was examined in rat heart mitochondria and in isolated cardiomyocytes or fibres which were permeabilized with the non-ionic detergent saponin to determine the kinetics of mitochondrial creatine kinase for beta-GPA. Fibres and myocytes were subjected to increasing [ADP] in the presence and absence of beta-GPA or creatine, whereas isolated mitochondria received a similar protocol with increasing [ATP]. In isolated mitochondria given ATP, there was a stimulation of respiration by creatine, but no significant stimulation of respiration by beta-GPA. Further studies on fibres from control and beta-GPA-fed rats also found that beta-GPA is not utilized by the mitochondria, as evidenced by a lack of beta-GPA-stimulated respiration (Km for ADP = 142 +/- 23 microM) compared with control (Km for ADP from 161 +/- 23 microM), but no significant change in Vmax. Therefore the rat heart mitochondria are not responsive to beta-GPA as compared with creatine. Interestingly, the fibres from beta-GPA-fed rats had no creatine- or beta-GPA-stimulated respiration (Km for ADP = 57.3 +/- 7.2 microM for control, 54.2 +/- 7.2 microM with creatine, and 53.5 +/- 7.8 microM with beta-GPA). The mitochondria prepared from the hearts of rats exposed for 10 weeks to 1% beta-GPA in their diet had a significant decrease in Vmax. and a significant decrease in Km for ADP. Thus the hearts from beta-GPA-fed animals may be pathologic, due to a disruption of the creatine kinase energy circuit.

Non-Insulin and Non-Exercise Related Increase of Glucose Utilization in Rats and Mice.

The effects of high-energy phosphate contents in muscles on glucose tolerance and glucose uptake into tissues were studied in rats and mice. Enhanced glucose tolerance associated with depleted high-energy phosphates and elevated glycogen content in muscles and liver was observed in animals fed creatine analogue beta-guanidinopropionic acid (beta-GPA). Distribution of infused 2-[1-14C]deoxy-D-glucose in tissues especially in the soleus muscle, kidney, and brain was greater in mice fed beta-GPA than controls. The glucose uptake was decreased when the contents of ATP and glycogen were normalized following creatine supplementation. Plasma insulin in animals at rest was lower and its concentration after intraperitoneal glucose infusion tended to be less in animals fed beta-GPA than controls (p > 0.05), although the pattern of insulin response to glucose loading was similar to the control. The daily voluntary activity in beta-GPA fed mice was also less than controls. These results suggest that improved glucose tolerance is not related to elevated insulin concentration and/or decreased glycogen following exercise. Such improvement may be due to an increased mitochondrial energy metabolism caused by depletion of high-energy phosphates.

Adaptation of rat skeletal muscle to creatine depletion: AMP deaminase and AMP deamination.

AMP deaminase catalyzes deamination of the AMP formed in contracting muscles to inosine 5'-monophosphate (IMP). Slow-twitch muscle has only approximately 30% as high a level of AMP deaminase activity as fast-twitch muscle in the rat, and rates of IMP formation during intense contractile activity are much lower in slow-twitch muscle. We found that feeding the creatine analogue beta-guanidinopropionic acid (beta-GPA) to rats, which results in creatine depletion, causes a large decrease in muscle AMP deaminase. This adaptation was used to evaluate the role of AMP deaminase activity level in accounting for differences in IMP production in slow-twitch and fast-twitch muscles. beta-GPA feeding for 3 wk lowered AMP deaminase activity in fast-twitch epitrochlearis muscle to a level similar to that found in the normal slow-twitch soleus muscle but had no effect on the magnitude of the increase in IMP in response to intense contractile activity. Despite a similar decrease in ATP in the normal soleus and the epitrochlearis from beta-GPA-fed rats, the increase in IMP was only approximately 30% as great in the soleus in response to intense contractile activity. These results demonstrate that the accumulation of less IMP in slow- compared with fast-twitch skeletal muscle during contractile activity is not due to the lower level of AMP deaminase in slow-twitch muscle.

Linear dependence of muscle phosphocreatine kinetics on total creatine content.

Phosphorus nuclear magnetic resonance (NMR) spectra and twitch tension were recorded during stimulation of gastrocnemius muscles of pentobarbital sodium-anesthetized rats which had been fed the creatine analogue beta-guanidinopropionic acid (beta-GPA, 2% diet) for periods from 0 (control) to 8 wk. Total creatine content of unstimulated muscles decreased by 42, 67, 82, and 88% compared with controls after 2-, 4-, 6-, and 8-wk feeding, respectively. The staircase effect observed in control muscles during 8 min of twitch stimulation at 0.25, 0.5, and 0.75 Hz was reduced after 2- to 8-wk beta-GPA feeding. However, after 6- to 8-wk feeding, the twitch force at the end of 8 min of stimulation was not different from controls. The time constant for phosphocreatine (PCr) changes at the onset of and during recovery after stimulation was proportional to total creatine content. The relationship between PCr content and twitch rate times force at the end of stimulation was linear, with slope proportional to total creatine content. PCr content in beta-GPA-fed animals was transiently greater during recovery than before stimulation, suggesting a regulatory effect of the inorganic phosphate released by hydrolysis of phosphorylated beta-GPA. The results are consistent with linear models of respiratory control in which the creatine kinase reaction acts as a simple buffer of adenine nucleotide levels.