Β-guanidinopropionic Acid Research Articles

Microbial reconstitution reverses prenatal stress-induced cognitive impairment and synaptic deficits in rat offspring

Stress during pregnancy is often linked with increased incidents of neurodevelopmental disorders, including cognitive impairment. Here, we report that stress during pregnancy leads to alterations in the intestinal flora, which negatively affects the cognitive function of offspring. Cognitive impairment in stressed offspring can be reproduced by transplantation of cecal contents of stressed pregnant rats (ST) to normal pregnant rats. In addition, gut microbial dysbiosis results in an increase of β-guanidinopropionic acid in the blood, which leads to an activation of the adenosine monophosphate-activated protein kinase (AMPK) and signal transducer and activator of transcription 3 (STAT3) in the fetal brain. Moreover, β-guanidinopropionic acid supplementation in pregnant rats can reproduce pregnancy stress-induced enhanced glial differentiation of the fetal brain, resulting in impaired neural development. Using probiotics to reconstruct maternal microbiota can correct the cognitive impairment in the offspring of pregnant stressed rats. These findings suggest that microbial reconstitution reverses gestational stress-induced cognitive impairment and synaptic deficits in male rat offspring.

The futile creatine cycle and the synthesis of fatty acids in inguinal white adipose tissue from growing rats, submitted to a hypoprotein-hyperglycidic diet for 15 days.

The low-protein, high-carbohydrate (LPHC) diet administered to growing rats soon after weaning, for 15 days, promoted an increase in energy expenditure by uncoupling protein 1 (UCP1) in interscapular brown adipose tissue, and also due to the occurrence of the browning process in the perirenal white adipose tissue (periWAT). However, we believe that inguinal white adipose tissue (ingWAT) may also contribute to energy expenditure through other mechanisms. Therefore, the aim of this work is to investigate the presence of the futile creatine cycle, and the origin of lipids in ingWAT, since that tissue showed an increase in the lipids content in rats submitted to the LPHC diet for 15 days. We observed increases in creatine kinase and alkaline phosphatase activity in ingWAT, of the LPHC animals. The mitochondrial Nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide oxidized ratio is lower in ingWAT of LPHC animals. In the LPHC animals treated with β-guanidinopropionic acid, the extracellular uptake of creatine in ingWAT was lower, as was the rectal temperature. Regarding lipid metabolism, we observed that in ingWAT, lipolysis in vitro when stimulated with noradrenaline is lower, and there were no changes in baseline levels. In addition, increases in the activity of enzymes were also observed: malic, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase, in addition to an increase in the PPARγ content. The results show the occurrence of the futile creatine cycle in ingWAT, and that the increase in the relative mass may be due to an increase in de novo fatty acid synthesis.

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.

Suppressing Nonradiative Recombination in Lead–Tin Perovskite Solar Cells through Bulk and Surface Passivation to Reduce Open Circuit Voltage Losses

Lead–Tin perovskite solar cells (Pb/Sn PSCs) are limited by the intrinsic instability of Sn(II), which tends to oxidize forming Sn vacancies in perovskite films. Herein, a Lewis base β-guanidinopropionic acid (GUA) and hydrazinium iodide (HAI) are introduced to effectively passivate the perovskite bulk and surface, respectively. The synergistic approach leads to Pb/Sn PSCs with a promising power conversion efficiency of 20.5% owing to the significantly reduced nonradiative recombination and voltage losses. As a result, the VOC × FF product of PSCs is significantly improved, which is among the highest values documented in the literature, being favorable for perovskite-based tandem applications. Additionally, the strategy demonstrated in this work could also improve the stability of PSCs by enhancing the chemical robustness of the perovskite layer. These results emphasize the significance of bulk and surface passivation in the development of efficient and stable PSCs based on Pb/Sn perovskites.

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.

TAMI-25. UPREGULATION OF CREATINE METABOLISM BY MYELOID CELLS RESULTS IN GLIOBLASTOMA PROGRESSION

Abstract Malignant brain tumors are uniquely immunosuppressive, with a predominant infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs) and a deficit in T-cells unrivaled to any other tumor. This unique tumor microenvironment (TME) promotes resistance to both conventional and immune therapies for this disease. The underlying mechanisms by which TAMCs promote glioblastoma (GBM) progression are not fully understood. We found that TAMCs specifically upregulate de-novo creatine metabolism within GBM using unbiased genetic and metabolic screening. This metabolic phenotype was confirmed in human GBM patients by comparing peripheral versus tumor-infiltrating myeloid cells. Examination of de-novo creatine generation using Carbon13 arginine flux revealed that TAMCs, but not tumor-infiltrating CD8+ T-cells, can produce creatine. Furthermore, we demonstrate that TAMCs actively secrete de-novo generated creatine into cell cultures. Examination of the single-cell microenvironment of GBM revealed that malignant cells preferentially express the creatine transporter, indicating that TAMC-derived creatine is taken up by GBM. Notably, SLC6A8 is directly upregulated in the context of hypoxia and suggests that creatine uptake is a mechanism to promote survival under hypoxic stress. Indeed, exogenous creatine supplementation promoted both the migration and survival of multiple glioblastoma cell lines in-vitro. Utilizing an established inhibitor of creatine metabolism, β-Guanidinopropionic acid (β -GPA), we found that β -GPA blocks both the migration and survival of glioma cells under hypoxic stress. Lastly, β -GPA also inhibited creatine secretion by TAMCs, showing that creatine blockade can also influence TAMC metabolic phenotype. In the future, we will examine the importance of creatine metabolism on both immune suppression and tumor progression in-vivo. This work provides novel insights into the role of creatine metabolism in GBM and identifies a unique therapeutic avenue for this devastating disease.

Beta-guanidinopropionic acid has age-specific effects on markers of health and function in mice.

AMP-activated protein kinase (AMPK) is a central regulator of both lifespan and health across multiple model organisms. β-Guanidinopropionic acid (GPA) is an endogenous AMPK activator previously shown to improve metabolic function in young and obese mice. In this study, we tested whether age of administration significantly affects the physiological outcomes of GPA administration in mice. We report that intervention starting at 7-8 months (young) results in activation of AMPK signaling and a phenotype consisting of lower body mass, improved glucose control, enhanced exercise tolerance, and altered mitochondrial electron transport chain flux similar to previous reports. When GPA treatment is started at 18-19 months (old), the effect of GPA on AMPK signaling is blunted compared to younger mice despite similar accumulation of GPA in skeletal muscle. Even so, GPA administration in older animals delayed age-related declines in lean mass, improved measures of gait performance and circadian rhythm, and increased fat metabolism as measured by respiratory exchange ratio. These results are likely partially driven by the relative difference in basal function and metabolic plasticity between young and old mice. Our results suggest that age-related declines in AMPK sensitivity may limit potential strategies targeting AMPK signaling in older subjects and suggest that further research and development is required for AMPK activators to realize their full potential.

Graded 2D/3D Perovskite Heterostructure for Efficient and Operationally Stable MA-Free Perovskite Solar Cells.

Almost all highly efficient perovskite solar cells (PVSCs) with power conversion efficiencies (PCEs) of greater than 22% currently contain the thermally unstable methylammonium (MA) molecule. MA-free perovskites are an intrinsically more stable optoelectronic material for use in solar cells but compromise the performance of PVSCs with relatively large energy loss. Here, the open-circuit voltage (Voc ) deficit is circumvented by the incorporation of β-guanidinopropionic acid (β-GUA) molecules into an MA-free bulk perovskite, which facilitates the formation of quasi-2D structure with face-on orientation. The 2D/3D hybrid perovskites embed at the grain boundaries of the 3D bulk perovskites and are distributed through half the thickness of the film, which effectively passivates defects and minimizes energy loss of the PVSCs through reduced charge recombination rates and enhanced charge extraction efficiencies. A PCE of 22.2% (certified efficiency of 21.5%) is achieved and the operational stability of the MA-free PVSCs is improved.

A urinary metabolomic study from subjects after long-term occupational exposure to low concentration acrylamide using UPLC-QTOF/MS

Because long-term occupational exposure to low concentrations of acrylamide (ACR) has the potential to cause neurological damage, it is important to identify biomarkers that can be used to evaluate this risk. In the present study, urine metabolomics of the ACR-exposed and non-exposed groups to identify potential metabolites was carried out using ultra high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. Serum biochemical indexes of the exposed and non-exposed groups were also determined. Principal component analysis showed a differential separation between exposed group and non-exposed group and a total of 7 metabolites were identified in positive and negative ionization modes; Area under curve of anthranilic acid, β-guanidinopropionic acid and mesobilirubinogen were 0.980, 0.843 and 0.801 respectively and these metabolites showed high sensitivity and specificity. The 13 biochemical indexes were divided into three classes based on physiological functions. Only biomarkers of dysregulated liver function including alanine aminotransferase, aspartic transaminase, total bilirubin, direct bilirubin and triglyceride were significantly higher in the exposed group than in the non-exposed group. This study identifies important related metabolic changes in the bodies of workers after long-term occupational exposure to low concentration ACR and suggests new biomarkers of nervous system injury caused by ACR. The study also provides a sound basis for exploring the biochemical mechanisms and metabolic pathways of nervous system toxicity caused by ACR.

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.

Intracellular accumulation of amino acids increases synaptic potentials in rat hippocampal slices.

The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a transport system with a low affinity for taurine. The prototypic taurine transporter TauT (SLC6A6) was discarded by experimental evidence obtained in TauT-KO mice. The purpose of the present study was to determine whether the proton-coupled amino acid transporter 1 (PAT1; SLC36A1) which is a transport system with low affinity and high capacity for a great variety of amino acids including taurine, contributes to the taurine-induced synaptic potentiation. In rat hippocampal slices, the application of several amino acids (L- and D-alanine, L-glutamine, β-guanidinopropionic acid, glycine, L-histidine, L- and D-serine, sarcosine, L- and D-threonine) imitated the synaptic potentiation induced by taurine. The magnitude of the potentiation caused by some of these amino acids was even greater than that induced by taurine. By contrast, the application of other amino acids (L-arginine, betaine, L-leucine, L-methionine, L- and D-proline, and L-valine) did not induce potentiation. The behaviour of these different amino acids on synaptic potentiation is not compatible with a role of PAT1 in synaptic potentiation. There was a positive correlation between the accumulation of the different amino acids in the slice and the magnitude of synaptic potentiation induced by them. Some of the amino acids inducing synaptic potentiation, like taurine and L-threonine, also increased electrical resistance of the slice, whereas L-leucine did not modify this parameter. Modifications induced by either taurine or L-threonine in synaptic potentiation, slice resistance and amino acid accumulation were dependent on extracellular chloride concentration. These findings support the idea that the accumulation of amino acids throughout the action of transporters causes cell swelling enhancing the electrical resistance of the slice, which by itself could be sufficient to increase field synaptic potentials.

PO-104 The shifted balance of high-energy phosphates drives the AMPK dephosphorylation and reduced slow myosin expression during unloading

Objective Mechanical unloading of postural muscles results in both muscle atrophy and a slow to fast fibre type transition. The cause of such changes is a reduction in slow-type MyHC isoform expression and an increase in fast-type MyHC isoform expression. It is believed that calcineurin/NFAT signalling pathway and AMP-activated protein kinase (AMPK) are involved in the regulation of slow-type MyHC isoform. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading. We found, that stimulation of AMPK activity at the early stage of gravitational unloading prevents a decline of slow MyHC expression. We assumed that a decrease in AMPK activity in rat soleus at the early stage of gravitational unloading could be associated with changes in the ratio of intracellular high-energy phosphates (ATP/ADP). The aim of the study was to investigate the impact of high-energy phosphates ratio changes on AMPK activity and slow-type MyHC isoform expression in rat soleus muscle at the early stages of gravitational unloading.
 Methods To verify the hypothesis, we used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h hindlimb suspension. The content of phospho-AMPK, phospho-ACC, phospho-PKD, HDAC4/5 in rat soleus was determined by Western-blotting. The amount of MyHCI(β) pre-mRNA and mRNA was evaluated by RT-PCR. 
 Results After 24-h HS we observed a decrease (p<0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group. 24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control.
 Conclusions Thus βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates (ATP, ADP and creatine phosphate) takes place and leads to reduced AMPK activity and a slow to fast myosin fibre type transition. The study was supported by Russian Science Foundation grant # 18-15-00107.

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

Differential response of adipose tissue gene and protein expressions to 4- and 8-week administration of β-guanidinopropionic acid in mice.

β‐Guanidinopropionic acid (β‐GPA) feeding inhibits growth‐associated gain of body mass. It remains unknown, however, whether and how β‐GPA feeding affects growth‐associated increase in white adipose tissue (WAT) mass. We examined the effects of 4‐ and 8‐week β‐GPA feeding on serum myostatin levels and expression of genes and proteins related to adipogenesis, lipolysis, and liposynthesis in epididymal WAT (eWAT) and brown adipose tissue (BAT) in 3‐week‐old, juvenile male mice. Body, eWAT, and muscle weights were significantly lower in β‐GPA‐fed mice than in controls after feeding. Four‐ but not 8‐week‐β‐GPA feeding increased the serum myostatin level. Incubation of C2C12 myotubes with β‐GPA (1 mM) significantly promoted myostatin mRNA expression. The protein expression of peroxisome proliferator‐activated receptor gamma coactivator 1 α (PGC‐1α) and peroxisome proliferator‐activated receptor α (PPAR α) was up‐regulated in GPAF eWAT at week 4, but down‐regulated at week 8. There was no significant difference in the protein expression of adipocyte triglyceride lipase (ATGL), hormone‐sensitive lipase (HSL), fatty acid synthase (FAS), and acetyl‐CoA carboxylase (ACC) between groups in eWAT. In BAT, no significant difference was found in the protein expression of PGC‐1α, PPAR α, ATGL, and HSL between β‐GPA‐fed and control mice, whereas that of FAS and ACC was significantly lower in β‐GPA‐fed mice at week 8. Uncoupling protein 1 was expressed higher in β‐GPA‐fed mice both at weeks 4 and 8 than that in controls. Thus, the mechanism by which β‐GPA feeding in early juvenile mice inhibits growth‐associated increase in eWAT mass may differ between early and later periods of growth.

β-Guanidinopropionic Acid Stimulates Brain Mitochondria Biogenesis and Alters Cognitive Behavior in Nondiseased Mid-Age Mice

β-guanidinopropionic acid (β-GPA) has been used as a nutritional supplement for increasing physical strength and endurance with positive and predictable results. In muscles, it works as a nonadaptive stimulator of mitochondria biogenesis; it also increases lipid metabolism. There are data indicating that β-GPA can be also neuroprotective, but its mechanisms of action in the brain are less understood. We studied the effects of β-GPA on animal behavior and mitochondrial biogenesis in the cortex and midbrain of mid-age healthy mice. We found that even short-term 3-week-long β-GPA treatment increased the mitochondrial DNA (mtDNA) copy number in the cortex and ventral midbrain, as well as the expression of several key antioxidant and metabolic enzymes—indicators of mitochondria proliferation and the activation of Nrf2/ARE signaling cascade. At the same time, β-GPA downregulated the expression of the β-oxidation genes. Administration of β-GPA in mice for 3 weeks improved the animals’ physical strength and endurance health, ie, increased their physical strength and endurance and alleviated anxiety. Thus, β-GPA might be considered an adaptogene affecting both the muscle and brain metabolism in mammals.

The effects of dietary β-guanidinopropionic acid on growth and muscle fiber development in juvenile red porgy, Pagrus pagrus.

β-guanidinopropionic acid (β-GPA) has been used in mammalian models to reduce intracellular phosphocreatine (PCr) concentration, which in turn lowers the energetic state of cells. This leads to changes in signaling pathways that attempt to re-establish energetic homeostasis. Changes in those pathways elicit effects similar to those of exercise such as changes in body and muscle growth, metabolism, endurance and health. Generally, exercise effects are beneficial to fish health and aquaculture, but inducing exercise in fishes can be impractical. Therefore, this study evaluated the potential use of supplemental β-GPA to induce exercise-like effects in a rapidly growing juvenile teleost, the red porgy (Pagrus pagrus). We demonstrate for the first time that β-GPA can be transported into teleost muscle fibers and is phosphorylated, and that this perturbs the intracellular energetic state of the cells, although to a lesser degree than typically seen in mammals. β-GPA did not affect whole animal growth, nor did it influence skeletal muscle fiber size or myonuclear recruitment. There was, however, an increase in mitochondrial volume within myofibers in treated fish. GC/MS metabolomic analysis revealed shifts in amino acid composition of the musculature, putatively reflecting increases in connective tissue and decreases in protein synthesis that are associated with β-GPA treatment. These results suggest that β-GPA modestly affects fish muscle in a manner similar to that observed in mammals, and that β-GPA may have application to aquaculture by providing a more practical means of generating some of the beneficial effects of exercise in fishes.

β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy.

Previous studies have demonstrated that AMP-activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β-guanidinopropionic acid (β-GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β-GPA and aging remains elusive. In this study, we hypothesized that feeding β-GPA to adult Drosophila produces the lifespan extension via activation of AMPK-dependent autophagy. It was found that dietary administration of β-GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule-associated protein 1A/1B-light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β-GPA treatment, indicating that autophagic activity plays a role in the effect of β-GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β-GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β-GPA treatment significantly elevated the expression of phospho-T172-AMPK levels, while inhibition of AMPK by either AMPK-RNAi or compound C significantly attenuated the expression of autophagy-related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β-GPA can induce an extension of the lifespan of Drosophila via AMPK-Atg1-autophagy signaling pathway.

A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy.

Dietary supplementation of β-guanidinopropionic acid (βGPA) reduces whole-body and skeletal muscle growth in young CD-1 mice.

Increased AMP-activated protein kinase (AMPK) activity leads to enhanced fatty acid utilization, while also promoting increased ubiquitin-dependent proteolysis (UDP) in mammalian skeletal muscle. β-guanidinopropionic acid (βGPA) is a commercially available dietary supplement that has been shown to promote an AMPK-dependent increase in fatty acid utilization and aerobic capacity in mammals by compromising creatine kinase function. However, it remains unknown if continuous βGPA supplementation can negatively impact skeletal muscle growth in a rapidly growing juvenile. The current study was conducted to examine the effect of βGPA supplementation on whole-body and skeletal muscle growth in juvenile and young adult mice. Three-week old, post weanling CD-1 mice were fed a standard rodent chow that was supplemented with either 2% (w/w) α-cellulose (control) or βGPA. Control and βGPA-fed mice (n = 6) were sampled after 2, 4, and 8 weeks. Whole-body and hindlimb muscle masses were significantly (P < 0.05) reduced in βGPA-fed mice by 2 weeks. The level of AMPK (T172) phosphorylation increased significantly (P < 0.05) in the gastrocnemius of βGPA-fed versus control mice at 2 weeks, but was not significantly different at the 4- and 8-week time points. Further analysis revealed a significant (P < 0.05) increase in the skeletal muscle-specific ubiquitin ligase MAFbx/Atrogin-1 protein and total protein ubiquitination in the gastrocnemius of βGPA versus control mice at the 8-week time point. Our data indicate that feeding juvenile mice a βGPA-supplemented diet significantly reduced whole-body and skeletal muscle growth that was due, at least in part, to an AMPK-independent increase in UDP.

Contribution of the phosphagen system to postmortem muscle metabolism in AMP-activated protein kinase γ3 R200Q pig Longissimus muscle

Pigs with the AMP-activated protein kinase γ3 R200Q (AMPKγ3R200Q) mutation generate pork with low ultimate pH (pHu). We hypothesized that reducing muscle creatine (Cr) and phosphocreatine (PCr) may accelerate postmortem ATP consumption and prevent extended pH decline in AMPKγ3R200Q longissimus muscle. Wild type and AMPKγ3R200Q pigs were assigned to control diet or diet supplemented with the creatine analog β-guanidinopropionic acid (β-GPA, 1%) for 2wk. β-GPA reduced muscle PCr (P=0.006) and total Cr (P<0.0001). In general, AMPKγ3R200Q+β-GPA exhibited more rapid metabolism than control, AMPKγ3R200Q, and β-GPA treatment, evidenced by more rapid loss of ATP, more rapid increase in IMP, and decreased pH during the first 90min postmortem. Overall, pHu was similar despite elevated glycogen (AMPKγ3R200Q), reduced total Cr (β-GPA) or both (AMPKγ3R200Q+β-GPA). Thus, reducing muscle phosphagens did not affect pHu in AMPKγ3R200Q muscle, but it hastened ATP depletion and pH decline.