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Implications for lipid induced sarcopenia;Ishige okamurae and its active components prevent palmitate impaired skeletal myogenesis

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Sarcopenia, defined as the loss of muscle mass and strength with increasing age, leads to disability, frailty, morbidity, and mortality. Anorexia and cachexia lead to the loss of muscle mass. Besides decreased strength, the loss of muscle mass also negatively affects aerobic capacity and metabolic rate, ultimately reducing function and quality of life. Obesity, another health concern, is defined as abnormal and excessive body fat. In recent years, the prevalence of obese individuals in both middle and old age has increased. Similar to sarcopenia, the risk of obesity increases with age. The emerging and challenging epidemiological trends in developing countries include sarcopenia and obesity. The prevalence of obesity combined with sarcopenia is referred to as sarcopenic obesity/ obese-sarcopenia, which exhibits synergistic complications of both syndromes. Obesity causes substantial changes in fat metabolism, leading to fat deposition in nonadipose tissues such as skeletal tissue. The development of obesity is associated with high-lipid diets, predominantly saturated fatty acids such as palmitic acid (16:0, PA). PA is the most common saturated fatty acid in the human body. Excess fatty acid infiltration into skeletal tissues alters metabolic signaling. Moreover, growth molecular signaling and catabolic pathways that support the proper functioning of skeletal cells are also affected. Individuals with sarcopenic obesity have greater risks of metabolic disorders compared to those for sarcopenia or obesity alone. Strong and healthy muscles are vital for the handling of overload created by obesity. Skeletal muscle myogenesis is of the utmost importance in maintaining muscle mass and regenerative capacity. However, lipid overload disturbs skeletal myogenesis. vi The study evaluates association of PA in the myotubes formed via in vitro serum deprivation and the negative effects of fatty acids on skeletal muscle. The myocellular mechanisms in skeletal myogenesis are researched to understand its implications. Plant species that grow in the marine environment; namely, seaweeds or sea vegetables, have become an emerging sustainable nutritional source and an essential part of healthy human diets, mostly in the East Asian region. They are a good source of vitamins and minerals, fiber, and polysaccharides that support low caloric intake. Furthermore, seaweeds contain pigments, proteins, polyunsaturated fatty acids (PUFAs), and phenolic compounds. Polyphenols are an important component of marine algae (brown algae) that provide functional food and pharmaceutical significance. However, studies reporting their use as interventions in muscle-related research are limited. The study examined a non-cytotoxic concentration of PA to provide mechanistic insights into PA-impaired skeletal myogenesis and potential medicinal and dietary interventions through edible brown seaweed, Ishige okamurae (IO). C2C12 cells were examined for myogenic markers, adipogenic factors, and regenerative capacity through growth regulators against PA interference to assess IO and purified diphloroethohydoxycarmalol (DPHC) as potential treatments. Both IO and DPHC improved myogenic marker (myogenin, MyoD, and MyHC) levels. While PA down-regulated myogenic markers while improving adipogenic factors (PPARγ, c/EBPα, A-FABP), DPHC significantly arbitrated the negative effects. DPHC treatment also improved phosphorylation of the growth regulatory PI3K/Akt/mTOR axis over the adverse effects of PA. The results of this study suggested regulatory mechanisms through which the bioactive components IO and DPHC based on the sustainable utilization of I. okamurae inhibited the PA-induced impairment of skeletal myogenesis. vii Generation of reactive oxygen species (ROS) is closely related with the ectopic lipid accumulation due to improved flux of energy substrate such as excessive dietary fat, thus influence cellular dysfunction. Pathogenesis of diabetes and linked sarcopenia is associated with the activation of pathways related to oxidative stress and muscular mitochondrial dysfunction. Further, mitochondria function as a signaling platform for multiple biological responses including immunity and metabolism. Literature provides evidence on the function of mitochondrial ROS (mtROS) in the activation of nucleoside oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes and the regulation of innate signaling pathways. Controlled levels of mtROS are vital for the innate host defense against pathogens, though unrestrained amounts may lead to pathologies including chronic inflammation. The Nlrp3 inflammasome consisting of apoptotic speck-containing protein with a CARD (ASC) and inactive pro-caspase-1 conveys inflammatory signals downstream by cleaving pro-IL-1β to IL-1β. The secreted IL-1β and IL-18 delivers and continues inflammatory signaling. Palmitate influences the Nlrp3 inflammasome, mtROS as well as pattern recognition receptors (PRRs). Ultimately, this could lead to apoptosis which in myocellular environment is a tissue weakening. Experimental results demonstrate PA improved cell injury, where myotube injury was significantly attenuated by IO and DPHC. Moreover, PA promoted mtROS generation leading to Nlrp3 activation as well as transduction of NF-κB signaling. Notably, PA increased the Nlrp3 complex formation as evident by the immunofluorescence analysis while DPHC substantially abolished it. The PA-induced Nlrp3 activation was successfully reversed by MCC950, a Nlrp3 inhibitor to confirm the significance of Nlrp3 inflammasome formation in IL-1β and IL-18 release. This confirms the vital role of Nlrp3 inflammasome in the release of IL-1β and IL-18. Mito-Tempo, a mtROS scavenger viii remarkably reduced PA-induced Nlrp3 inflammasome activation. Additionally, PAstimulated production of IL-1β and IL-18 were as well inhibited with the Mito-Tempo treatment. Taken together, the results implicate mtROS production induced by PA alters cellular dynamics and plays a central role in the Nlrp3 inflammasome mediated IL-1β and IL-18 expression. Collectively, DPHC is a potent alleviator of mtROS where it reverses mitochondrial dynamics thus providing mitigation of inflammasome activation. Further into the research, ishphloroglucin A (IPA), another active component from IO exhibited skeletal muscle regenerative potential. Palmitate induced skeletal myogenesis effects were hampered by the compound where it also substantially inhibited the Nlrp3 driven skeletal dysfunction. A better understanding and of the molecular pathways of lipid-induced sarcopenia can have profound implications in the development of therapeutic interventions. Thus, the results of this study suggest the potential for treatments based on the bioactive components IO, DPHC, and IPA with the sustainable utilization of the I. okamurae marine algae.
Jayawardena Thilina Uduwaka
Issued Date
Awarded Date
2022. 2
Alternative Author(s)
자야와데나 틸리나 우두와카
제주대학교 대학원
대학원 해양생명과학과
Table Of Contents
Part Ⅰ: Background and significance 1
1.1 Introduction. 2
1.2 Muscle atrophy 7
1.2.1 Importance of skeletal muscle, mass, and function 7
1.2.2 Obese-sarcopenia 14
1.2.3 Ageing related changes in muscle structure and function 15
1.2.4 Molecular pathways involved in sarcopenia 24
1.3 Proteolytic systems in skeletal muscle 28
1.3.1 Ubiquitin-proteasome system. 28
1.3.2 Caspase-mediated proteolysis (need to construct from the beginning) 30
1.3.3 Autophagy-lysosome system 31
1.4 The role of seafood as a preventive measurement for sarcopenia 35
1.5 Conclusions. 47
1.6 Goal of the study . 48
References. 49
Part Ⅱ: Ishige okamurae, diphloroethohydroxycarmalol, and ishophloroglucin A inhibit palmitic acid-impaired skeletal myogenesis and improve muscle regenerative potential . 60
2.1 Introduction. 61
2.2 Material and methods 64
2.2.1 Reagents . 64
2.2.2 Obtaining 70% ethanol extract from Ishige okamurae. 64
2.2.3 The isolated compounds from Ishige okamurae. 65
2.2.4 Muscle cell culture and myotube formation. 67
2.2.5 Experimental treatments. 67
2.2.6 Determining optimal palmitate and compound concentrations 67
2.2.7 Western blotting . 68
2.2.8 Immunofluorescence 69
2.2.9 RT-qPCR 69
2.2.10 Statistical analysis 70
2.3 Results. 71
2.3.1 Potential of IO extract 71
2.3.2 Palmitate inhibits skeletal muscle differentiation and myogenic regulatory factor expression 73
2.3.3 Effect of palmitate on myoblast proliferation 73
2.3.4 Palmitate affects skeletal muscle differentiation 75
2.3.5 DPHC, IPA effects on myoblast and myotubes . 80
2.3.6 DPHC, IPA, and PA behavior in myotubes . 83
2.3.7 Palmitate improves adipogenic character in muscle cells 86
2.3.8 Effects of palmitate, DPHC, and IPA on the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) growth regulatory axis . 89
2.3.9 IPA ameliorates atrophic signaling in PA-induced myotube weakening. 92
2.4 Discussion . 94
2.5 Conclusion. 101
References. 102
Part Ⅲ: Mitochondrial ROS/Nlrp3 inflammasome axis contribute PA-induced skeletal muscle wasting and Ishige okamurae purified DPHC as a potent intervention 106
3.1 Introduction. 107
3.2 Material and methods 110
3.2.1 Reagents . 110
3.2.2 Cell culture . 110
3.2.3 Experimental treatments. 111
3.2.4 Determining optimal palmitate and compound concentrations 111
3.2.5 Analysis of mitochondrial membrane potential by flowcytometer 111
3.2.6 ROS detection. 112
3.2.7 Immunofluorescence 112
3.2.8 Western blot analysis 113
3.2.9 ELISA detection . 113
3.2.10 Statistical analysis 113
3.3 Results. 115
3.3.1 Palmitate affects myoblast cell viability. 115
3.3.2 Effect of IO extract and DPHC on myoblast survival 115
3.3.3 IO extract inhibits PA induced ROS generation. 118
3.3.4 Palmitate induced mitochondrial damage/depolarization is weakened by DPHC, lleviating mitochondrial ROS production 121
3.3.5 DPHC alleviated the expression and formation of Nlrp3 inflammasome in C2C12 myotubes stimulated by PA 124
3.3.6 Palmitate activates Nlrp3 inflammasome through mitochondrial ROS production 129
3.3.7 DPHC inhibit PA-induced nuclear translocation of NF-κB. 133
3.3.8 DPHC improves PA-altered antioxidant enzyme pathway proteins 136
3.3.9 DPHC inhibits PA-induced caspase-3/caspase-9 activation and restore the PAaltered ax/Bcl-2 balance 138
3.3.10 Palmitate leads to muscle wasting in-vitro by influencing on atrogenes . 140
3.4 Discussion . 142
3.5 Conclusions. 146
References. 148
Concluding remarks. 151
Acknowledgment. 152
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