The World Health Organization (WHO) estimates that chronic inflammatory conditions accounts for more than 17 million deaths every year [1
]. Obesity is considered one of the main underlying factors for chronic inflammation and has almost doubled since 1980 and now over 1.4 billion adults worldwide are obese. Obesity, once considered a problem only in developed countries, is now also a major problem in low- and middle-income countries. Parallel to the increase in obesity, diseases associated with chronic inflammation such as cardiovascular diseases and type 2 diabetes are also rising [1
]. One of the major concerns associated with low-grade inflammation is sarcopenia—loss of muscle mass. Generally, after the age of 50 the muscle mass reduces at a rate of up to 1%–2% annually, while fat mass increases [2
]. At the same time, muscle strength drops at the rate of 1.5% annually between the age of 50 and 60 years and at the rate of 3% thereafter [4
]. Obesity accelerates age-related muscle deterioration [5
] exacerbating the risk for type 2 diabetes (T2D) by approximately 30% [6
]. The negative feedback loop defined as the combination of excess weight-gain and reduced muscle mass, strength and performance is known as sarcopenic obesity [7
]. Further suggestions of a role for inflammation in sarcopenia includes an association between increased TNF-α [8
] and Il-6 [9
] and decreased muscle mass and strength.
Skeletal muscle (SkM) constitutes about 40% of total body mass in adult lean men and is an adaptable tissue in response to changes in lifestyle such as diet and physical training [10
]. SkM is one of the major sites of glucose metabolism, accounting for about 30% of postprandial glucose disposal [12
]. This makes SkM a crucial organ for maintaining healthy glucose concentrations in the body, with loss of muscle mass also associated with increased type 2 diabetes risk. Changes to diet may impact on SkM and use of fish oil rich in the fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been proposed to improve SkM metabolism [13
]. An intervention with marine fatty acids improved muscle mass and function in older adults [13
] and animal studies suggest a role for marine fatty acids in SkM anabolism and increased protein synthesis [14
]. In addition, marine fatty acids have anti-inflammatory activity in animals and humans [16
], which may reduce the loss of inflammation-mediated SkM mass in older adults. Supplementation with EPA and DHA, the two main marine fatty acids, could be the basis for a simple, safe and low-cost solution for preventing and mitigating negative changes to SkM metabolism. For such a strategy to be credible, it is crucial to understand the underlying signaling mechanisms mediated by EPA and DHA on SkM.
Our previous intervention studies showed that mice fed high fat diets (HFDs) supplemented with herring improve muscle mass compared with mice fed HFD supplemented with beef [17
]. In several randomized control trials in humans, marine fatty acids improve muscle volume, a surrogate marker for improved muscle performance [13
]. In order to build on these results and determine if EPA and DHA, or other components in fatty fish, are responsible for improving muscle metabolism and to determine what the underlying mechanisms could be, we conducted a follow-up trial on earlier work with fatty fish [17
]. Here we have used transcriptomics to explore how replacing a commonly used source of dietary fat, corn oil with purified EPA and DHA interacts with gastrocnemicus skeletal muscle (gSkM) gene expression, in order to investigate how EPA and DHA can affect muscle deterioration.
In this study, we have shown that HFD-ED improves muscle metabolism and promotes a switch to slow-fiber type compared to HFD-corn oil. HFD-ED improves metabolism of fatty acids in gSkM by upregulating expression of genes coding for proteins involved in fatty acid β-oxidation, the Krebs cycle and oxidative phosphorylation. Lipid analysis shows that the HFD-ED fed animals had lower total fatty acid content compared to HFD-corn oil and control diet fed animals. Reduced ectopic fat accumulation in HFD-ED compared with HFD-corn oil fed animals suggests a physiological effect of EPA and DHA on muscle metabolic health. Higher expression of the genes involved in muscle contraction was found in gSkM of HFD-ED fed animals compared with HFD-corn oil fed animals. Western blot analysis showed reduced Acc protein levels in gSkM of HFD-ED fed animals compared with HFD-corn oil, confirming gene expression results. Acc is an inhibitor of fatty acid β-oxidation in mitochondria and the lower levels shows increase in mitochondrial fatty acid β-oxidation. In addition, increased Tnnc1 levels suggest increased muscle contraction in gSkM of HFD-ED fed animals compared with HFD-corn oil. Moreover, increased nuclear protein levels of the Nfatc4 isoform strongly suggests up-regulation of slow-fiber-type gene expression in gSkM of HFD-ED fed animals compared with HFD-corn oil fed animals (Figure 4
The gSkM neutral lipid and free fatty acid profiles reflect the higher amount of DHA in the feed of HFD-ED fed mice, though EPA was not detected in either fraction, in line with our previous findings in liver; that EPA was present in very low amounts, not in proportion to EPA in the diet [21
]. There was no substantial proportional difference in gSkM fatty acid composition between the two HFD groups, even though HFD-corn oil fed mice had nearly twice the amount of total neutral lipids. This suggests that ED does not specifically upregulate any of the reactions related to β-oxidation and rather, leads to stimulation of β-oxidation in general, resulting in reduced total neutral lipids in SkM. Although there were several differences in the amount and proportion of free fatty acids and phospholipids, these differences were not quantitatively important aside from the increase DPA and DHA in mice fed HFD-ED but lower EPA in phospholipids fractions [33
]. Notably C18:1 was substantially higher in HFD-corn oil fed mice compared with HFD-ED fed mice and this result was reversed for DHA. This could have implications for membrane fluidity within SkM and this may play a role in the observed transcriptome changes.
Previously we found that HFD-ED induced β-oxidation reduced liver fat compared to HFD-corn oil [21
], similarly, reduced SkM fat can be due to the increase in β-oxidation and the lower amount of neutral lipids in HFD-ED fed mice. Reduced SkM mitochondrial fatty acid β-oxidation has been proposed as a key feature leading to insulin resistance [34
] and high fat diets led to downregulation of the group of genes involved in oxidative phosphorylation in human SkM biopsies [36
]. In the gSkM of the HFD-ED fed mice, the expression of rate limiting enzymes in fatty acid β-oxidation in mitochondria, Cpt1
were upregulated compared with HFD-corn oil fed mice. Notably, the protein level of Acc was significantly reduced in gSkM of HFD-ED fed mice compared with HFD-corn oil. Acc regulates fatty acid metabolism by synthesizing malonyl-CoA (building blocks for new fatty acids) but suppresses mitochondrial β-oxidation by inhibiting fatty acid transporter Cpt1
. Therefore, lower protein levels of Acc suggests entry of fatty acid into mitochondria via Cpt1
transporter for mitochondrial β-oxidation in gSkM of HFD-ED fed mice compared with HFD-corn oil. A SkM specific isoform of Acc
) knockout mice were protected against fat-induced peripheral insulin resistance [39
] whereas overexpression of Cpt1
is sufficient to reduce insulin resistance [40
]. Impairments in mitochondrial oxidative capacity in skeletal muscle have been seen in relation to a decrease in energy expenditure [35
]. Thus, increased mitochondrial oxidative capacity in the gSkM of HFD-ED fed animals, supported by increased levels by Cpt1
and decreased total protein levels of Acc may contribute to increased energy expenditure. Further work on a possible stimulation of energy expenditure by EPA and DHA, possibly via diet-induced thermogenesis is required.
Ageing and obesity may contribute to the decrease in the contractibility of skeletal muscle. Muscle contraction is regulated by intracellular calcium concentration via the thin filament regulatory proteins troponin and tropomyosin. In the absence of calcium, actin-myosin interaction and subsequently muscle contraction is inhibited. In the gSkM of HFD-ED, two ATPases, namely Na+
-ATPase transporting α2 polypeptide (Atp1a2
; sodium pump) and ATPase Ca+2
transporting skeletal muscle slow switch 2 (Atp2a2
) were upregulated compared with HFD-corn oil. A tissue specific knockout study shows that the sodium pump (Atp1a2
) is required to prevent fatigue, and systematic analysis of Serca isoforms in calcium transport showed their importance for restoring muscle contractibility [43
]. Protein levels of the oxidative fiber biomarker Troponin C1 were higher in gSkM of HFD-ED fed mice compared with HFD-corn oil, indicative of increased muscle contraction via control of intracellular calcium in mice fed HFD-ED. Together, these results suggest that HFD-ED contributes to increased calcium release into the sarcoplasm, compared with HFD-corn oil fed mice.
Calcineurin has been proposed to play a major role in the upregulation of slow-fiber-specific gene expression [45
]. Earlier, it was found that inhibition of calcineurin by cyclosporin-A could result in an increased number of fast fibers in rat skeletal muscle [45
]. Activated calcineurin phosphatase capacity is required for dephosphorylation of Nfat proteins, which then localize to the nucleus to initiate slow-fiber-specific gene expression [45
]. Our data shows that the gSkM of HFD-ED fed animals has markedly higher nuclear levels of Nfat (Nfatc4 isoform) compared with HFD-corn oil, which could contribute to upregulation of the slow-fiber-specific gene expression upon nuclear localization. An earlier study showed abundant myogenin mRNA in slow oxidative muscles and this relationship followed phenotype transition caused by cross-innervation [48
]. In HFD-ED fed mice, myogenin (Padj
-value = 0.06) tends to be higher compared with HFD-corn oil fed animals suggesting upregulation of genes for the slower muscle fiber phenotype. We also found that EPA and DHA increased gene expression of Ppargc1
α, a master regulator of mitochondrial biogenesis leading to red type I fiber, also suggests genetic programing towards stimulating slower muscle fiber [49
]. It is known that the products of peroxisomal β-oxidation are also targeted towards mitochondria for oxidation in skeletal muscle and therefore assist in complete lipid disposal [53
]. Further support for an effect on muscle fiber type comes from stimulation of insulin growth factor 1 (Igf
-1), which regulates the Akt
pathway. Activation of Akt
β thereby inactivating it. Gsk3
β phosphorylates Nfat
and excluding its entry into the nucleus and subsequent DNA binding [54
], which would down-regulate slow-fiber gene expression. In our study, Gsk3
β was downregulated in the gSkM of HFD-ED compared with HFD-corn oil animals, indicating little Nfat phosphorylation and thus, conditions that would favor production of slow-fiber type. Resistance training, testosterone treatment, growth hormone, or dehydroepiandrosterone interventions have shown favorable effects in retaining muscle mass and function [19
]. However, long-term use of growth hormone treatment can be harmful due to associated side effects, making this a poor choice for intervention against muscle loss. Our findings suggest a mechanistic basis for the human results and supports the case for further research on either ED or fish oil supplements as potential molecules for the long-term prevention of sarcopenia. In this study we were unable to determine if muscle fiber type regulation is due to the effects of increased β-oxidation, or a direct effect of EPA or DHA on gene transcription.
Although the effects of EPA and DHA on gSkM gene expression are conclusive, due to the limited sample amount we have been unable to validate many of the possible physiological effects found from transcriptome analysis. Ideally, future work should study these effects across a wider range of SkM and perform histology to confirm the hypothesis on fiber-type regulation. Similarly, our results suggest an effect on many parameters related to energy metabolism, supported in part by finding a lower fat content in the gSkM of HFD-ED fed mice compared with HFD-corn oil. Future work will need to see if this effect extends to improved glucose disposal which could extend the findings to possible relevance for T2D. A role for regulation of calcium concentrations for signaling should also be tested using muscle cell models. Future work will also need to test if these pathways are still relatively upregulated with lower proportions of n-3 fatty acids and at lower amounts, to test if they are relevant at normal amounts of dietary fat.
These results clearly demonstrate the wide-ranging impact a difference in dietary fatty acid composition can have on SkM gene-transcription. It is unclear if ED regulates all of these gene targets simultaneously, or if some are a result of downstream interactions with initial ED targets. Given the clear sequential effects on energy metabolism from β-oxidation to oxidative phosphorylation, it appears likely that the observed upregulation of these processes is directly related to increased circulating EPA and DHA. Further studies on cell and rodent models of ageing and functional analysis of SkM are required to confirm these effects and the likely regulatory nodes for these mechanisms.