scholarly journals The effect of young and old ex vivo human serum on cellular protein synthesis and growth in an in vitro model of ageing

2021 ◽  
Author(s):  
Sophie L. Allen ◽  
Ryan N. Marshall ◽  
Sophie J Edwards ◽  
Janet M. Lord ◽  
Gareth G. Lavery ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Human Serum ◽  
Signaling Pathways ◽  
Ex Vivo ◽  
Muscle Protein ◽  
C2c12 Cells ◽  
Muscle Loss ◽  
Older Individuals ◽  
Age Related

In vitro models of muscle ageing are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from 4 old (72 ± 1 years) and 4 young (26 ± 3 years) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1-hour and conditioned with human serum (10%) for 4 or 24-hours. After 4-hours C2C12 cells were treated with 5mM leucine for 30-minutes. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways measured via Western Blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared to young serum prior to leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.

scholarly journals Regulation of muscle protein synthesis in an in vitro cell model using ex vivo human serum

2018 ◽  
Vol 103 (6) ◽  
pp. 783-789 ◽  
Author(s):  
Brian P. Carson ◽  
Bijal Patel ◽  
Miryam Amigo-Benavent ◽  
Martina Pauk ◽  
Sunil Kumar Gujulla ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Human Serum ◽  
Cell Model ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Protein Synthesis

scholarly journals Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

2017 ◽  
Vol 312 (1) ◽  
pp. E27-E36 ◽  
Author(s):  
Servane Le Plénier ◽  
Arthur Goron ◽  
Athanassia Sotiropoulos ◽  
Eliane Archambault ◽  
Chantal Guihenneuc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Fold Increase ◽  
Underlying Mechanism ◽  
Muscle Homeostasis

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated ( P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

scholarly journals A cell-based evaluation of a non-essential amino acid formulation as a non-bioactive control for activation and stimulation of muscle protein synthesis using ex vivo human serum

PLoS ONE ◽  
2019 ◽  
Vol 14 (11) ◽  
pp. e0220757
Author(s):  
Bijal Patel ◽  
Martina Pauk ◽  
Miryam Amigo-Benavent ◽  
Alice B. Nongonierma ◽  
Richard J. Fitzgerald ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Human Serum ◽  
Essential Amino Acid ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Acid Formulation ◽  
A Cell ◽  
Stimulation Of

scholarly journals A cell-based evaluation of a non-essential amino acid formulation as a non-bioactive control for activation and stimulation of muscle protein synthesis using ex vivo human serum

2019 ◽  
Author(s):  
Bijal Patel ◽  
Martina Pauk ◽  
Miryam Amigo-Benavent ◽  
Alice B. Nongonierma ◽  
Richard J. Fitzgerald ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Human Serum ◽  
Ex Vivo ◽  
Muscle Protein ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Effective Control ◽  
C2c12 Myotubes

AbstractPurposeThe purpose of this study was to compare the effect of treating skeletal muscle cells with media conditioned by postprandial ex vivo human serum fed with either isonitrogenous NEAA or a whey protein hydrolysate (WPH) on stimulating MPS in C2C12 skeletal muscle cells.MethodsBlood was taken from six young healthy males following overnight fast (fasted) and 60 min postprandial (fed) ingestion of either WPH or NEAA (0.33 g.kg-1 Body Mass). C2C12 myotubes were treated with media conditioned by ex vivo human serum (20%) for 4 h. Activation of MPS signalling (phosphorylation of mTOR, P70S6K and 4E-BP1) were determined in vitro by Western Blot and subsequent de novo MPS were determined in vitro by Western Blot and surface sensing of translation technique (SUnSET) techniques, respectively.ResultsMedia conditioned by NEAA fed serum had no effect on protein signalling or MPS compared to fasted, whereas media conditioned by WPH fed serum significantly increased mTOR, P70S6K and 4E-BP1 phosphorylation (p<0.01, p<0.05) compared to fasted serum. Furthermore, the effect of media conditioned by WPH fed serum on protein signalling and MPS was significantly increased (p<0.01, p<0.05) compared to NEAA fed serum.ConclusionIn summary, media conditioned by NEAA fed serum did not result in activation of MPS. Therefore, these in vitro findings suggest the use of isonitrogenous NEAA acts as an effective control for comparing bioactivity of different proteins on activation of MPS. These findings also confirm that activation of MPS in C2C12 myotubes treated with media conditioned by WPH-fed serum is primarily due to circulating EAA.

scholarly journals Casein Protein Hydrolysate Fed Human Serum From Older Adults Regulates In Vitro Muscle Protein Synthesis, Muscle Protein Breakdown and Myotube Growth

2020 ◽  
Author(s):  
Martina Pauk ◽  
Miryam Amigo-Benavent ◽  
Bijal Patel ◽  
Philip M. Jakeman ◽  
Brian P. Carson
Keyword(s):  
Older Adults ◽  
Protein Synthesis ◽  
De Novo ◽  
Muscle Protein ◽  
Protein Hydrolysate ◽  
Muscle Protein Synthesis ◽  
Protein Breakdown ◽  
Age Related ◽  
Casein Protein

In this study we used a recently developed ex vivo-in vitro model to assess the effect of feeding older adults a casein protein hydrolysate (CPH) compared with non-bioactive non-essential amino acid (NEAA) supplement on Muscle Protein Synthesis (MPS) and Breakdown (MPB). Serum from six healthy older males following overnight fast and 60 min postprandial ingestion of CPH or NEAA (0.33 g.kg-1 body mass) was used to condition C2C12 myotube media. CPH-fed serum significantly increased MPS compared to fasted serum. In addition, CPH-fed serum induced myotube growth and markedly suppressed atrogin-1, but not MuRF1, expression. Comparatively, no change in MPS, myotube growth and gene expression was observed following NEAA-fed serum treatment. CPH-fed serum from older adults stimulated de novo MPS, suppressed markers of protein breakdown and resulted in myotube growth, indicating a potential role for CPH as a dietary protein source to prevent age-related sarcopenia.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

scholarly journals Advances in the Role of Leucine-Sensing in the Regulation of Protein Synthesis in Aging Skeletal Muscle

2021 ◽  
Vol 9 ◽  
Author(s):  
Yan Zhao ◽  
Jason Cholewa ◽  
Huayu Shang ◽  
Yueqin Yang ◽  
Xiaomin Ding ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Therapeutic Potential ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Trna Synthetase ◽  
Limiting Factor ◽  
Anabolic Resistance ◽  
Age Related

Skeletal muscle anabolic resistance (i.e., the decrease in muscle protein synthesis (MPS) in response to anabolic stimuli such as amino acids and exercise) has been identified as a major cause of age-related sarcopenia, to which blunted nutrition-sensing contributes. In recent years, it has been suggested that a leucine sensor may function as a rate-limiting factor in skeletal MPS via small-molecule GTPase. Leucine-sensing and response may therefore have important therapeutic potential in the steady regulation of protein metabolism in aging skeletal muscle. This paper systematically summarizes the three critical processes involved in the leucine-sensing and response process: (1) How the coincidence detector mammalian target of rapamycin complex 1 localizes on the surface of lysosome and how its crucial upstream regulators Rheb and RagB/RagD interact to modulate the leucine response; (2) how complexes such as Ragulator, GATOR, FLCN, and TSC control the nucleotide loading state of Rheb and RagB/RagD to modulate their functional activity; and (3) how the identified leucine sensor leucyl-tRNA synthetase (LARS) and stress response protein 2 (Sestrin2) participate in the leucine-sensing process and the activation of RagB/RagD. Finally, we discuss the potential mechanistic role of exercise and its interactions with leucine-sensing and anabolic responses.

scholarly journals Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion

2020 ◽  
Vol 21 (10) ◽  
pp. 3631 ◽  
Author(s):  
Raffaella Boggia ◽  
Federica Turrini ◽  
Alessandra Roggeri ◽  
Guendalina Olivero ◽  
Francesca Cisani ◽  
...  
Keyword(s):  
Oral Administration ◽  
Ellagic Acid ◽  
Ex Vivo ◽  
Behavioral Skills ◽  
Aged Mice ◽  
Age Related ◽  
The Central Nervous System ◽  
The Impact ◽  
Old Mice

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in “ex-vivo, in vitro” parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

scholarly journals Antioxidant Role of PRGF on RPE Cells after Blue Light Insult as a Therapy for Neurodegenerative Diseases

2020 ◽  
Vol 21 (3) ◽  
pp. 1021 ◽  
Author(s):  
Carlota Suárez-Barrio ◽  
Susana del Olmo-Aguado ◽  
Eva García-Pérez ◽  
María de la Fuente ◽  
Francisco Muruzabal ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Cell Line ◽  
Antioxidant Effect ◽  
Age Related Macular Degeneration ◽  
Strong Impact ◽  
Retinal Diseases ◽  
Age Related ◽  
Gene And Protein Expression

Oxidative stress has a strong impact on the development of retinal diseases such as age-related macular degeneration (AMD). Plasma rich in growth factors (PRGF) is a novel therapeutic approach in ophthalmological pathologies. The aim of this study was to analyze the antioxidant effect of PRGF in retinal epithelial cells (EPR) in in vitro and ex vivo retinal phototoxicity models. In vitro analyses were performed on ARPE19 human cell line. Viability and mitochondrial status were assessed in order to test the primary effects of PRGF. GSH level, and protein and gene expression of the main antioxidant pathway (Keap1, Nrf2, GCL, HO-1, and NQO1) were also studied. Ex vivo analyses were performed on rat RPE, and HO-1 and Nrf2 gene and protein expression were evaluated. The results show that PRGF reduces light insult by stimulating the cell response against oxidative damage and modulates the antioxidant pathway. We conclude that PRGF’s protective effect could prove useful as a new therapy for treating neurodegenerative disorders such as AMD.

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