Obesity causes an increase in macrophage infiltration into tissues that are the targets of insulin signaling including adipose, liver and muscle (reviewed in [1
]). We reported that macrophage co-culture with human myotubes blunts insulin signaling and augments atrophy signaling pathways, and these effects are exacerbated by palmitic acid, a saturated FFA that is inflammatory [6
]. In this study, the same co-culture system was used to compare the effect of unsaturated FFAs, oleic acid and DHA, to that of palmitic acid on insulin and atrophy signaling pathways in myotubes. Macrophage co-culture dramatically decreased myotube pAkt levels, and DHA did not protect against this. The major effects of either palmitic acid or DHA were to reduce baseline pAkt levels, and there was no effect of either macrophages or FFAs on the relative increase in pAkt due to insulin stimulation. This is surprising, given that ceramide and DAG levels were increased. Atrophy signaling pathways were induced by both macrophage co-culture and palmitic acid which increased MAFbx mRNA levels and Fn14 protein levels. The addition of DHA reduced Fn14 protein levels in a statistically significant manner and there was a trend to reduce MuRF-1 protein levels. Finally, DHA treatment likely induced a post-translational modification of MuRF-1 suggesting a possible mechanism to target MuRF-1 for degradation. Thus, DHA may have a beneficial effect on human muscle mass by reducing MuRF-1 ubiquitin ligase levels.
Suppression of baseline pAkt levels by DHA was unexpected, but its mechanism is likely to be different than palmitic acid. DHA has been shown to increase PTEN levels [25
] and inhibit PI3K [26
], both of which could reduce pAkt levels. Alternatively, in adipocytes, DHA increases pAkt levels by a pathway that involves the heterotrimeric G-protein coupled receptor GPR120 [15
]. However, in mice this receptor is specifically expressed in cell types that include adipocytes and macrophages, but not muscle [15
]. Thus, DHA treatment either increases or decreases pAkt depending upon the cell type. The precise mechanism that is induced by DHA treatment to regulate pAkt levels in human myotubes remains to be elucidated.
Although reducing baseline pAkt, palmitic acid treatment did not reduce the magnitude of the stimulation of pAkt levels in response to insulin. Whereas FFAs are known to induce insulin resistance in humans [5
], there have been reports that cultured myotubes can be resistant to palmitic acid treatment. Bikman and colleagues reported that pooled myotubes from obese individuals were not inhibited by a 16 h pretreatment with 0.45 mM palmitic acid, a higher concentration than used in this study [27
]. Alternatively, Thrush and colleagues, using isolated muscle preparations, found that muscle from lean but not obese humans was resistant to the effects of 2 mM palmitic acid [28
]. We confirmed that palmitic acid increased DAG and ceramide levels. However, further investigation will be necessary to determine the mechanism for resistance to palmitic acid treatment.
Because of the remarkable reduction in pAkt in myotubes co-cultured with macrophages and our previous work [6
], we also evaluated atrophy signaling. Macrophages are required for many aspects of muscle biology such as repair [29
] and hypertrophy responses [30
]. Depending on their activation state, macrophages have dramatically different effects on muscle including damage [31
], induction of atrophy signaling [6
], modulation of insulin signaling [6
], and supporting satellite cell proliferation and repair [29
]. Both M1 and M2 macrophages are found in human muscle, with M2 primarily responding to hypertrophic stimuli from resistance training [34
]. We hypothesize that obesity results in increased abundance of classically activated macrophages in muscle, and that this contributes to insulin resistance. The THP-1 monocytes used in this study were activated by PMA treatment which induces a classical activation state that is inflammatory and induces atrophy signaling [6
]. Co-culture with these macrophages results in a dramatic reduction of pAkt which would be predicted to induce both MAFbx and MuRF-1 since activation of Akt can dominantly repress both of these ubiquitin ligases [35
]. Furthermore, macrophage co-culture also increased Fn14. Fn14 is a receptor for TWEAK (TNF-like weak inducer of apoptosis), and Fn14-TWEAK signaling is thought to play a role in tissue repair [36
]. In muscle, Fn14 is induced after denervation; whereas, TWEAK remains constant, indicating an “inside-out” signaling mechanism in which receptor levels are critical [23
]. Furthermore, the analysis of TWEAK-Tg and TWEAK-null mice suggests that TWEAK-Fn14 signaling induces atrophy in mice by inducing MuRF-1 and that disruption of TWEAK-Fn14 signaling could have beneficial effects on muscle mass [23
]. The observation in this study that macrophage co-culture induces Fn14 suggests that MuRF-1 should increase. Whereas MAFbx mRNA was induced by macrophage co-culture at 24 hours, MuRF-1 mRNA was not induced. This suggests that the kinetics of induction of MAFbx and MuRF-1 in myotubes in response to macrophage co-culture are different. This could be due to a differential response to the pAkt-FOXO pathway and the Fn14-TWEAK pathway, both of which are induced by macrophage co-culture.
Omega-3 fatty acids such as DHA are anti-inflammatory and use multiple biochemical mechanisms to down-regulate inflammatory signaling pathways in cells [15
]. Although the macrophages used in this study were activated towards an inflammatory state, the effect that they had on human muscle pAkt could not be disrupted by DHA treatment. However, the atrophy-inducing signaling by macrophage co-culture could be disrupted by DHA since Fn14 was not up-regulated by macrophage co-culture in the presence of DHA. Furthermore, DHA treatment reduced MuRF-1 protein levels in the majority of the cell lines tested. Atrophy occurs with aging and certain diseases such as cancer and diabetes [39
]. There have been reports that fish oil supplementation protects against weight loss in cancer [40
], and that reduced plasma omega-3 fatty acid levels are associated with muscle loss [42
]. Finally, omega-3 fatty acid supplementation was shown to increase grip strength in the elderly, suggesting that fish oil supplementation preserves muscle mass in humans [43
]. Thus, the ability of DHA to inhibit Fn14 signaling by reducing Fn14 protein levels and the ability of DHA to reduce MuRF-1 protein levels suggest that DHA treatment induces multiple mechanisms of lowering MuRF-1 protein levels and that DHA dietary supplementation may preserve muscle mass in humans.
Finally, DHA treatment caused a decrease in the mobility of MuRF-1, suggesting that MuRF-1 is post-translationally modified in myotubes. Since MuRF-1 protein levels tended to be reduced by DHA treatment, and phosphorylation is the first step in directing a number of proteins to the ubiquitin-proteasome degradation pathway, we determined whether phosphorylation caused the decrease in the mobility of Murf-1. Dephosphorylation of the protein extract with lambda phosphatase did not increase the electrophoretic mobility of MuRF-1, but was effective since pAkt was dephosphorylated (). Thus, phosphorylation is not responsible for the reduced electrophoretic mobility of MuRF-1 observed in response to DHA treatment. The modification may be acylation, perhaps by DHA itself, although palmitic acid and oleic acid treatment did not affect MuRF-1electrophoretic mobility. Thus, the identity of the post-translational modification and whether it causes MuRF-1 to be a substrate for ubiquitin ligases remain to be determined.