Creatine Analogue Research Articles

Oral β-GPA induces daily torpor in the mouse

In response to caloric deficit and a cool environment, mice (Mus musculus Linnaeus, 1758) employ daily torpor, a physiological state characterized by metabolic rate depression and bradycardia, followed by a decrease in body temperature. Although some heat is generated during arousal from torpor via uncoupling-protein 1 (UCP1)-dependent activity, there is also evidence of UCP1-independent mechanisms. We hypothesized that mice utilize futile creatine cycling as a UCP1-independent mechanism for generating heat during arousal from torpor, and thus interference with this cycle would result in longer arousal times. β-guanidinopropionic acid (β-GPA), a creatine analog shown to decrease cellular creatine levels, was used to assess the role of the futile creatine cycle in arousal from torpor. Mice consuming a standard 1% β-GPA diet lost body weight and, unexpectedly, experienced drops in body temperature that appear “torpor-like” in nature. Behavioral (body position) and physiological (body temperature dynamics, heart rate dynamics and variability, surface tail temperature) assessments were used to distinguish between torpor-like bouts induced by 1% β-GPA feeding and natural torpor. Our analysis suggests that the torpor-like bouts induced during feeding of 1% β-GPA should be considered daily torpor. Contrary to our initial hypothesis, the rate of arousal from these bouts was not significantly affected by β-GPA treatment. Still, the utilization of torpor by mice in a fed state is an unusual finding, and one of the few cases where food consumption does not inhibit daily torpor in the mouse

Exogenous Beta-guanidinopropionic acid administration enhances electromyostimulation-induced mitochondrial biogenesis in rat skeletal muscle

Abstract Objectives Exercise training induces several skeletal muscle adaptations. Beta-guanidinopropionic acid (β-GPA) is a creatine analog that simulates the effect of exercise to induce mitochondrial biogenesis. However, the effects of β-GPA on resistance training adaptation, such as muscle hypertrophy and mitochondrial biogenesis, are unclear. Therefore, using a resistance exercise model in rats, the present study was designed to investigate the effects of β-GPA administration on resistance training adaptations. Methods This study was approved by the Ethics Committee for Animal Experiments at Ritsumeikan University (approval number: BKC2022-009). Male Sprague Dawley rats were randomly divided into placebo or β-GPA groups. β-GPA (1000 mg/kg) was orally administered once daily, starting seven days before the initiation of electromyostimulation as a model for resistance exercise, and continued throughout the training period. Electromyostimulation was applied to the right gastrocnemius muscle via electrical stimulation every other day for a total of 12 sessions Results Peroxisome proliferators-activated receptor-γ co-activator-1α, a regulator of mitochondrial biogenesis, was significantly increased by the combination of training and β-GPA compared to the training leg (p<0.05). Protein expression of Total OXPHOS, a marker of mitochondrial content, was significantly increased by the combination of training and β-GPA compared to the training leg (p<0.05). β-GPA intake reduced muscle mass (main effect of β-GPA, p<0.05) and was associated with muscle protein breakdown-related Fbx32 and LC3-II protein expression levels but did not counteract the increase in muscle mass caused by resistance training. Conclusions Administration of exogenous β-GPA enhanced resistance training-induced mitochondrial biogenesis. Moreover, β-GPA still permitted resistance electromyostimulation-induced muscle mass gains, but that effect was attenuated as compared to placebo.

Ucp1 Ablation Improves Skeletal Muscle Glycolytic Function in Aging Mice.

Muscular atrophy is among the systematic decline in organ functions in aging, while defective thermogenic fat functionality precedes these anomalies. The potential crosstalk between adipose tissue and muscle during aging is poorly understood. In this study, it is showed that UCP1 knockout (KO) mice characterized deteriorated brown adipose tissue (BAT) function in aging, yet their glucose homeostasis is sustained and energy expenditure is increased, possibly compensated by improved inguinal adipose tissue (iWAT) and muscle functionality compared to age-matched WT mice. To understand the potential crosstalk, RNA-seq and metabolomic analysis were performed on adipose tissue and muscle in aging mice and revealed that creatine levels are increased both in iWAT and muscle of UCP1 KO mice. Interestingly, molecular analysis and metabolite tracing revealed that creatine biosynthesis is increased in iWAT while creatine uptake is increased in muscle in UCP1 KO mice, suggesting creatine transportation from iWAT to muscle. Importantly, creatine analog β-GPA abolished the differences in muscle functions between aging WT and UCP1 KO mice, while UCP1 inhibitor α-CD improved muscle glycolytic function and glucose metabolism in aging mice. Overall, these results suggested that iWAT and skeletal muscle compensate for declined BAT function during aging via creatine metabolism to sustain metabolic homeostasis.

Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.

Increased plasma creatine concentrations are associated with the risk of type 2 diabetes, but whether this alteration is associated with or causal for impairments in metabolism remains unexplored. Because skeletal muscle is the main disposal site of both creatine and glucose, we investigated the role of intramuscular creatine metabolism in the pathophysiology of insulin resistance in type 2 diabetes. In men with type 2 diabetes, plasma creatine concentrations were increased, and intramuscular phosphocreatine content was reduced. These alterations were coupled to reduced expression of sarcomeric mitochondrial creatine kinase 2 (CKMT2). In C57BL/6 mice fed a high-fat diet, neither supplementation with creatine for 2 weeks nor treatment with the creatine analog β-GPA for 1 week induced changes in glucose tolerance, suggesting that increased circulating creatine was associated with insulin resistance rather than causing it. In C2C12 myotubes, silencing Ckmt2 using small interfering RNA reduced mitochondrial respiration, membrane potential, and glucose oxidation. Electroporation-mediated overexpression of Ckmt2 in skeletal muscle of high-fat diet-fed male mice increased mitochondrial respiration, independent of creatine availability. Given that overexpression of Ckmt2 improved mitochondrial function, we explored whether exercise regulates CKMT2 expression. Analysis of public data revealed that CKMT2 content was up-regulated by exercise training in both humans and mice. We reveal a previously underappreciated role of CKMT2 in mitochondrial homeostasis beyond its function for creatine phosphorylation, independent of insulin action. Collectively, our data provide functional evidence for how CKMT2 mediates mitochondrial dysfunction associated with type 2 diabetes.

TMET-14. TUMOR-ASSOCIATED MYELOID CELL-DERIVED CREATINE PROMOTES GLIOBLASTOMA GROWTH IN HYPOXIC NICHES AND PSEUDOPALISADING TUMOR REGIONS

Abstract Glioblastoma (GBM) consists of a unique tumor microenvironment (TME) dominated by infiltrating tumor-associated myeloid cells (TAMCs). TAMCs are central to tumor growth and understanding how they support tumor progression is critical to identifying new therapeutic modalities. Examination of the metabolic and genetic phenotypes of TAMCs revealed that the de-novo creatine phenotype is central to their identity. Immunohistochemistry and multiplex fluorescence in GBM patient-derived tissue showed TAMC infiltration localizing to hypoxic pseudopalisading regions. Furthermore, single-cell RNAseq analysis of human and mouse models of GBM identified that the transporter of creatine, Slc6a8, is located on tumor cells within these regions. Spatial transcriptomics confirmed this transporter-synthesis gene compartmentalization is specific to hypoxic pseudopalisading regions, and further elucidated a radial glial cell gene signature for tumor cells expressing Slc6a8. Murine models recapitulated the TAMC de-novo creatine metabolic phenotype both ex-vivo and in-vitro. Metabolomics showed that TAMC-derived creatine can transfer to tumor cells in-vitro and is enhanced under hypoxic conditions. β-guanidinopropionic acid (β-GPA) is a creatine analog proposed to block Slc6a8 function and effectively blocked TAMC-derived creatine transfer to tumor cells in-vitro. Furthermore, β-GPA inhibited both the viability and size of spheres for tumor cells grown in stem-cell conditions. Lastly, inhibition of creatine transport using clinically relevant inhibitors enhanced survival following tumor implantation in murine models. This work highlights the key role of creatine in the pseudopalisading and hypoxic niche in GBM, offering evidence for a potential benefit in targeting this axis for GBM therapy regimens.

Age- and time-dependent mitochondrial genotoxic and myopathic effects of beta-guanidinopropionic acid, a creatine analog, on rodent skeletal muscles.

Beta-guanidinopropionic acid (GPA) is a creatine analog suggested as a treatment for hypertension, diabetes, and obesity, which manifest primarily in older adults. A notable side effect of GPA is the induction of mitochondrial DNA deletion mutations. We hypothesized that mtDNA deletions contribute to muscle aging and used the mutation promoting effect of GPA to examine the impact of mtDNA deletions on muscles with differential vulnerability to aging. Rats were treated with GPA for up to 4months starting at 14 or 30months of age. We examined quadriceps and adductor longus muscles as the quadriceps exhibits profound age-induced deterioration, while adductor longus is maintained. GPA decreased body and muscle mass and mtDNA copy number while increasing mtDNA deletion frequency. The interactions between age and GPA treatment observed in the quadriceps were not observed in the adductor longus. GPA had negative mitochondrial effects in as little as 4weeks. GPA treatment exacerbated mtDNA deletions and muscle aging phenotypes in the quadriceps, an age-sensitive muscle, while the adductor longus was spared. GPA has been proposed for use in age-associated diseases, yet the pharmacodynamics of GPA differ with age and include the detrimental induction of mtDNA deletions, a mitochondrial genotoxic stress that is pronounced in muscles that are most vulnerable to aging. Further research is needed to determine if the proposed benefits of GPA on hypertension, diabetes, and obesity outweigh the detrimental mitochondrial and myopathic side effects.

Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression.

Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

Stimulation of autophagy and synaptic maintenance are commonalities induced by an exercise‐mimetic and diet supplement to avoid initiators of age‐related cognitive decline

Brain aging causes synaptic and cellular vulnerabilities, leading to cognitive impairment and an increased risk for dementia. Removal of old and damaged proteins becomes less efficient with age, causing protein accumulation stress and corresponding synaptopathy as protein clearance systems fail. Interestingly, physical exercise and a healthy diet are well known to promote cognitive health, perhaps by delaying or reducing age‐related changes that cause synaptic compromise. It is vital to understand the mechanism by which exercise and diet positively influence brain health since they are the only avenues implicated in reducing dementia risk. Accordingly, we tested if an exercise‐mimetic and a dietary supplement have a common influence on the protein clearing autophagy‐lysosomal pathway, and if such a mechanistic link promotes synaptic maintenance and resilience. Our study used long‐term brain explants that exhibit multi‐neural cell types and subfields, as well as native neuronal circuitries and synaptic density as compared to the intact adult brain. The exercise‐mimetic compound β‐guanidinopropionic acid (β‐GPA), a creatine analog, and a specific extract of American ginseng (P. quinquefolius; USANA Health Sci), were applied daily to the matured, 3‐dimensional cultures of hippocampal tissue for 3‐6 days. The gently harvested explants were then assessed by immunoblot for several markers including the cathepsin B (CatB) component of the autophagy‐lysosomal pathway, a protease found to improve clearance of pathogenic proteins and preserve synaptic and cognitive measures in models of AD, α‐synucleinopathy, and mild cognitive impairment (Mueller‐Steiner et al. 2006 ‐ Neuron 51:703; Butler et al. 2011 ‐ PLoS ONE 6:e20501; Hwang et al. 2019 ‐ Inter J Mol Sci 20:4432). Both β‐GPA and the American ginseng extract improved the levels of the active CatB isoform and increased autophagic flux. In addition, they both had a positive influence on synaptic markers. Next, β‐GPA and American ginseng were tested in groups of explant cultures that were exposed to a lysosomal inhibitor (chloroquine) as a model for the age‐related compromise of lysosomal protein clearance capacity. Again, both β‐GPA‐ and American ginseng‐treated hippocampal explants displayed protection of pre‐ and postsynaptic proteins, similar to the protected levels produced through pharmacological enhancement of CatB using small‐molecule modulators with drug‐like properties (Viswanathan et al. 2012 – ACS Med Chem Lett 3:920). The significant results from the chloroquine model indicate that preventing age‐related lysosomal stress can slow or delay the associated synaptopathy by different strategies that may be additive. Together, the different experiments support the idea that exercise, exercise‐mimetics, and certain natural products help to limit age‐related synaptic deterioration that is known to be the best correlate of cognitive failure during aging and dementia. The common protective influences mediated by the exercise‐mimetic and the dietary supplement point to protein clearance as having a mechanistic role for healthy cognitive aging.

Selected Articles from This Issue

Glioblastoma multiforme (GBM) is a highly aggressive cancer characterized by drug resistance and invasion. While it has been recognized that gap junctions (GJ) between GBM tumor cells and adjacent astrocytes are important for GBM invasion, the molecular mechanisms by which GJ contribute to GBM cell invasion have yet to be described. In their study, McCutcheon and Spray query the role of GJ protein Connexin 43 (Cx43) in GBM cell invasion via Cx43 genetic ablation and pharmacological inhibition in GBM cell line-astrocyte co-cultures and ex vivo brain organotypic slice cultures. The authors reveal a dichotomous function of Cx43 in GBM cell invasion, where Cx43 inhibition in GBM cells enhances GBM cell invasion and Cx43 inhibition in astrocytes abrogates GBM cell invasion. Transcriptomics analysis implicated miR-19b in the mechanism underlying the role of Cx43 in GBM cell invasion, an implication that was confirmed both in vitro and ex vivo. Overall, the study unveils a previously unknown mechanism governing GBM cell invasion that could inform targeted GBM therapeutic strategies moving forward.While dysregulated Notch signaling has been implicated in multiple cancer types, the role(s) of MIB1, an E3 ubiquitin ligase that is key for Notch signaling, in cancer has yet to be defined. Wang and colleagues show that MIB1 expression is increased in some lung squamous carcinomas and lung adenocarcinomas relative to adjacent healthy tissues, and that elevated MIB1 expression correlates with poor patient survival. Using a stably transduced MIB1 overexpressing cell line, the authors demonstrate that MIB1 can induce epithelial–mesenchymal transition and tumor cell motility. Conversely, eliminating MIB1 via CRISPR/Cas9 induced a more epithelial phenotyp. characterized by reduced tumor cell motility. The authors also find that MIB1 renders lung cancer cells susceptible to death induced by glutathione peroxidase 4 inhibitor RSL3, suggesting MIB1 expression sensitizes tumor cells to ferroptosis. Correspondingly, the authors show that MIB1 ubiquitinates master antioxidant transcription factor NRF2, allowing lipid peroxide accumulation and subsequent ferroptosis. Altogether, the data presented elucidate how MIB1 can contribute to tumorigenesis and suggest that patients bearing tumors with elevated MIB1 expression may benefit from treatment with ferroptosis-inducing agents.FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are prevalent in acute myeloid leukemia (AML) and are associated with poor prognoses. Existing FLT3 inhibitors can induce drug resistance and lose efficacy against AML, making combinatorial therapeutic approaches against FLT3-ITD–mutant AML an area of interest. By querying transcriptomes of FLT3-ITD–mutant AML cell lines treated with FLT3 inhibitors, Zhang and colleagues observed that FLT3-ITD mutations correlate with increased expression of de novo creatine biosynthesis pathway components. Accordingly, the authors found that FLT3-ITD–mutant AML cells contain heightened levels of creatine and ATP. The authors discovered that FLT3-ITD mutations increase de novo creatine biosynthesis in part via STAT5-mediated transcription of glycine amidinotransferase (AGAT), a critical enzyme in the creatine biosynthetic pathway. A creatine analog and shRNA-mediated AGAT ablation increased FLT3-ITD–mutant AML cell apoptosis and decreased proliferation, and each approach synergized with FLT3 inhibition to impede AML cell proliferation and survival in vitro. Overall, the study elucidates a previously unknown mechanism by which FLT3-ITD mutations promote AML and provides a new therapeutic target for patients with AML harboring FLT3-ITD mutations.Human papillomaviruses (HPV) are causally linked to several cancers, but molecular host–pathogen interactions underlying oncogenesis have not been fully elucidated. In their study, Mirza and colleagues show that Ecdysoneless (ECD), the human homolog of the Drosophila ecdysoneless gene, is highly expressed in head and neck squamous cell carcinoma cell lines as well as cervical cancer cell lines and tumor samples. Using in vitro protein–protein binding and cellular immunoprecipitation assays, the authors demonstrate that ECD binds to high-risk HPV E6 oncoprotein. Ablating ECD expression using siRNA revealed that ECD participates in E6 RNA splicing, as well as splicing of cellular mRNA encoding proteins that are critical for HPV-driven oncogenesis. Disruption of ECD expression also abrogated cervical cancer cell line colony formation, migration, and invasion, further supporting the finding that ECD contributes to HPV-driven oncogenesis. Taken together, the presented data further clarify molecular HPV-host interactions underlying HPV-driven oncogenesis and provide a potential novel therapeutic target for HPV-positive cancers.

HIF-Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy.

Simple SummaryTargeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents.The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 μg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 μg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

Diagnostic and Pharmacological Potency of Creatine in Post-Viral Fatigue Syndrome.

Post-viral fatigue syndrome (PVFS) is a widespread chronic neurological disease with no definite etiological factor(s), no actual diagnostic test, and no approved pharmacological treatment, therapy, or cure. Among other features, PVFS could be accompanied by various irregularities in creatine metabolism, perturbing either tissue levels of creatine in the brain, the rates of phosphocreatine resynthesis in the skeletal muscle, or the concentrations of the enzyme creatine kinase in the blood. Furthermore, supplemental creatine and related guanidino compounds appear to impact both patient- and clinician-reported outcomes in syndromes and maladies with chronic fatigue. This paper critically overviews the most common disturbances in creatine metabolism in various PVFS populations, summarizes human trials on dietary creatine and creatine analogs in the syndrome, and discusses new frontiers and open questions for using creatine in a post-COVID-19 world.

Myosin Heavy Chain Composition, Creatine Analogues, and the Relationship of Muscle Creatine Content and Fast-Twitch Proportion to Wilks Coefficient in Powerlifters.

Machek, SB, Hwang, PS, Cardaci, TD, Wilburn, DT, Bagley, JR, Blake, DT, Galpin, AJ, and Willoughby, DS. Myosin heavy chain composition, creatine analogues, and the relationship of muscle creatine content and fast-twitch proportion to Wilks coefficient in powerlifters. J Strength Cond Res 34(11): 3022-3030, 2020-Little data exist on powerlifting-specific skeletal muscle adaptations, and none elucidate sex differences in powerlifters. Powerlifters tend to display higher fast-twitch fiber content and phosphagen system dependence. Nevertheless, it is unknown whether fast-twitch fiber or muscle creatine content are predictive of competitive powerlifting performance (via Wilks coefficient). Twelve actively competing powerlifters (PL; n = 6M/6F; age = 21.3 ± 1.0; 3.0 ± 1.8 year competing; 7.3 ± 6.6 meets attended) and 10 sedentary controls (CON; n = 5M/5F; age = 19.4 ± 2.0 year) underwent vastus lateralis muscle biopsies and venipuncture to compare the myosin heavy chain (MHC) fiber type and creatine analogue profiles between groups of both sexes, and determine whether MHC IIa and muscle total creatine (MTC) composition predict powerlifting performance. Samples were analyzed for specific MHC isoform (I, IIa, and IIx) content via mixed homogenate SDS-PAGE, and creatine analogues (MTC, muscle creatine transporter [SLC6A8], serum total creatine [STC], and serum creatinine [CRT]). Furthermore, MHC IIa and MTC content were compared with Wilks coefficient using Pearson correlation coefficients. Male PL MHC content was 50 ± 6% I, 45 ± 6% IIa, and 5 ± 11% IIx, versus 46 ± 6% I, 53 ± 6 IIa, and 0% IIx in female PL. Conversely, male CON MHC content was 33 ± 5% I, 38 ± 7% IIa, and 30 ± 8% IIx, vs. 35 ± 9% I, 44 ± 8% IIa, and 21 ± 17% IIx in female CON. Muscle total creatine, SLC6A8, STC, and CRT did not significantly differ between groups nor sexes. Finally, neither MHC IIa content (r = -0.288; p = 0.364) nor MTC (r = 0.488; p = 0.108) significantly predicted Wilks coefficient, suggesting these characteristics alone do not determine powerlifting skill variation.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 μg/mL; AUC0-24 of 1970 h*μg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Cyclocreatine Transport by SLC6A8, the Creatine Transporter, in HEK293 Cells, a Human Blood-Brain Barrier Model Cell, and CCDSs Patient-Derived Fibroblasts.

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. Cells were incubated at 37°C with [14C]cyclocreatine (9μM) and [14C]creatine (9μM) 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. [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. 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.

Longitudinal metabolomics in dried bloodspots yields profiles informing newborn screening for succinic semialdehyde dehydrogenase deficiency.

Analyses of 19 amino acids, 38 acylcarnitines, and 3 creatine analogues (https://clir.mayo.edu) were implemented to test the hypothesis that succinic semialdehyde dehydrogenase deficiency (SSADHD) could be identified in dried bloodspots (DBS) using currently available newborn screening methodology. The study population included 17 post‐newborn SSADHD DBS (age range 0.8‐38 years; median, 8.2 years; 10 M; controls, 129‐353 age‐matched individuals, mixed gender) and 10 newborn SSADHD DBS (including first and second screens from 3 of 7 patients). Low (informative) markers in post‐newborn DBS included C2‐ and C4‐OH carnitines, ornithine, histidine and creatine, with no gender differences. For newborn DBS, informative markers included C2‐, C3‐, C4‐ and C4‐OH carnitines, creatine and ornithine. Of these, only creatine demonstrated a significant change with age, revealing an approximate 4‐fold decrease. We conclude that quantitation of short‐chain acylcarnitines, creatine, and ornithine provides a newborn DBS profile with potential as a first tier screening tool for early detection of SSADHD. This first tier evaluation can be readily verified using a previously described second tier liquid chromatography‐tandem mass spectrometry method for γ‐hydroxybutyric acid in the same DBS. More extensive evaluation of this first/second tier screening approach is needed in a larger population.

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. MethodsStable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. ResultsInhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. General significanceThese studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

β-guanidinopropionic acid and metformin differentially impact autophagy, mitochondria and cellular morphology in developing C2C12 muscle cells.

The serine/threonine kinase AMP-activated protein kinase (AMPK) is a drug target for the treatment of obesity and type 2 diabetes (T2D). Metformin, a widely prescribed anti-hyperglycemic agent, and β-guanidinopropionic acid (β-GPA), a dietary supplement and creatine analog, have been shown to increase activity of AMPK. Macroautophagy is an intracellular degradation pathway for aggregated proteins and dysfunctional organelles, which can be mediated by AMPK. The present study sought to elucidate how metformin and β-GPA affect cell morphology, AMPK activity, autophagy and mitochondrial morphology and function in developing C2C12 myotubes. β-GPA reduced myotube diameter and increased length throughout differentiation, while metformin increased myotube diameter only at the 48 h time point. β-GPA treatment enhanced AMPK signaling and expression of autophagy-related proteins. β-GPA treatment also increased the density of autophagosomes, autolysosomes, and lysosomes. Metformin also increased activation of AMPK after 48 h, but in contrast to β-GPA, led to a dramatic reduction in the density of autophagosomes and lysosomes. Both metformin and β-GPA reduced the mitochondrial oxygen consumption rate, and differentially altered mitochondrial morphology. Obesity and T2D have been shown to increase mitochondrial dysfunction and reduce autophagic flux in skeletal muscle cells. Therefore, β-GPA may help to alleviate the effects of metabolic disease by increasing autophagic flux in skeletal muscle cells. In contrast, the reduction of autophagy by metformin may lead to dysregulation of mitochondrial maintenance, as well as muscle development.

Phosphocyclocreatine is the dominant form of cyclocreatine in control and creatine transporter deficiency patient fibroblasts.

Creatine transporter deficiency (CTD) is a metabolic disorder resulting in cognitive, motor, and behavioral deficits. Cyclocreatine (cCr), a creatine analog, has been explored as a therapeutic strategy for the treatment of CTD. We developed a rapid, selective, and accurate HILIC ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method to simultaneously quantify the intracellular concentrations of cCr, creatine (Cr), creatine‐d3 (Cr‐d3), phosphocyclocreatine (pcCr), and phosphocreatine (pCr). Using HILIC‐UPLC‐MS/MS, we measured cCr and Cr‐d3 uptake and their conversion to the phosphorylated forms in primary human control and CTD fibroblasts. Altogether, the data demonstrate that cCr enters cells and its dominant intracellular form is pcCr in both control and CTD patient cells. Therefore, cCr may replace creatine as a therapeutic strategy for the treatment of CTD.

Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway

How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth isincompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2+ breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2+ patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.