Docosahexaenoic Acid Prevents the Activation of Proteolytic Signaling by Palmitate in Skeletal Muscle Cells: Implications for Lipid-Induced Muscle Atrophy Pubblico
Woodworth-Hobbs, Myra Ellen (2014)
Published
Abstract
Skeletal muscle atrophy is a comorbidity common to numerous chronic illnesses and is an independent prognostic indicator of mortality in these patients. Some of these illnesses are characterized by dyslipidemia and the ectopic accumulation of lipids in skeletal muscle. Accumulation of saturated lipids such as palmitate in skeletal muscle causes derangements in insulin-related signaling and other cellular insults such as endoplasmic reticulum (ER) stress which augment the activities of multiple proteolytic systems. The latter responses result in wasting or atrophy. Notably, high doses of fish oil and combinations of omega-3 polyunsaturated fatty acids (PUFAs) have been found to help cancer patients maintain lean body mass. In other studies, PUFAs, including docosahexaenoic acid (DHA), have been shown to positively regulate some signaling pathways that control protein homeostasis in skeletal muscle. These findings led us to hypothesize that DHA counteracts palmitate-induced atrophy of cultured muscle cells by preventing the dysregulation of various proteolytic systems and their upstream signaling pathways. This dissertation focuses on elucidating the mechanism(s) by which DHA positively impacts muscle protein metabolism by examining the effects of palmitate and DHA on Akt- and ER stress-related signaling pathways in an in vitro model system. The protein kinase Akt is a key modulator of protein balance that inhibits the forkhead box O (FoxO) transcription factors which selectively induce the transcription of atrophy-inducing genes in the ubiquitin-proteasome and autophagy-lysosome systems. ER stress induces the unfolded protein response which causes perturbations in protein turnover and mitochondrial instability, leading to activation of autophagy and caspase-mediated proteolysis. Our work demonstrates that DHA prevents the suppression of Akt activity and induction of ER stress induced by palmitate, thus limiting the up-regulation of the ubiquitin-proteasome, autophagy-lysosome, and caspase-mediated proteolytic systems caused by the saturated fatty acid. The experiments presented in this dissertation are the first to directly examine the effects of DHA on protein catabolism in cultured muscle cells. The results establish a rationale for further research into the use of DHA as a treatment to counteract the loss of muscle mass due to catabolic illnesses.
Table of Contents
CHAPTER 1. 1
INTRODUCTION. 1
CHAPTER 2. 5
BACKGROUND AND SIGNIFICANCE. 5
Skeletal muscle atrophy. 6
Overview of skeletal muscle. 6
Insulin resistance and type 2 diabetes mellitus. 9
Muscle atrophy and diabetes. 10
Proteolytic systems in skeletal muscle. 13
Ubiquitin-proteasome system. 14
Caspase-mediated proteolysis. 20
Autophagy-lysosome system. 21
Calpains. 27
Regulation of proteolysis in skeletal muscle. 27
Endoplasmic reticulum stress and the unfolded protein response. 30
The ATF6 pathway. 32
The IRE1a-XBP1 pathway. 34
The PERK-eIF2a-ATF4 pathway. 34
The PERK-Nrf2 pathway. 36
ER associated degradation. 37
CHOP mediates ER stress-induced cell death. 38
Endoplasmic reticulum stress and UPR is activated in disease. 41
Fatty Acids. 42
Fatty acid structure and nomenclature. 42
Synthesis of saturated and omega-3 polyunsaturated fatty acids. 44
Efficiency of omega-3 conversion in humans. 47
Dietary consumption of saturated and omega-3 fatty acids. 48
Relationship of palmitate and omega-3 fatty acids and cardiovascular health. 50
Potential cellular mechanisms underlying the differential effects of saturated and unsaturated fatty acids. 51
Lipids and skeletal muscle. 54
Dietary lipids affect skeletal muscle lipids. 54
Lipids and skeletal muscle insulin resistance. 55
Lipids and ER stress. 59
Lipids and muscle atrophy. 63
Summary and Significance. 70
CHAPTER 3. 73
DOCOSAHEXAENOIC ACID PREVENTS PALMITATE-INDUCED ACTIVATION OF PROTEOLYTIC SYSTEMS IN C2C12 MYOTUBES. 73
Introduction. 74
Methods and Materials. 76
Cultured myotube model. 76
Experimental treatments. 77
Protein degradation assay. 77
RNA isolation and qPCR analysis. 78
Isolation of cytosolic and nuclear cell fractions. 78
Western blot analysis. 79
Statistical analyses. 80
Results. 80
DHA attenuates the effects of palmitate on protein degradation. 80
DHA attenuates palmitate-induced inhibition of Akt-FoxO3 signaling. 80
DHA attenuates the effects of palmitate on proteolytic systems. 84
Discussion. 87
Acknowledgements. 94
CHAPTER 4. 95
DOCOSAHEXAENOIC ACID COUNTERACTS PALMITATE-INDUCED ER STRESS IN C2C12 MYOTUBES: INSIGHTS INTO FATTY ACID-INDUCED MUSCLE ATROPHY. 95
Background. 96
Methods and Materials. 98
Cultured myotube model. 98
Experimental treatments. 99
RNA isolation and qPCR analysis. 99
Western blot analysis. 99
Statistical analyses. 100
Results. 100
DHA attenuates the induction of ER stress by palmitate. 100
DHA prevents palmitate-induced activation of ER stress-associated proteolytic pathways. 102
Discussion. 108
CHAPTER 5. 115
DISCUSSION AND CONCLUSIONS. 115
Fatty acids affect muscle protein balance. 116
Akt/FoxO signaling and ER stress collaboratively regulate proteolysis in myotubes. 117
Future directions. 120
The potential role of protein synthesis in palmitate-induced myotube atrophy. 120
The potential role of oxidative stress in myotube atrophy: differential effects of palmitate and DHA. 121
DHA may prevent disruption of ER calcium homeostasis. 123
Palmitate and DHA may differentially alter microRNA expression. 124
Conclusions. 125
REFERENCES. 127
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