Skeletal muscle is the most widely distributed tissue in the body, which is involved in many important biological functions. It is also an important site to regulate the whole-body metabolism. However, pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 may cause muscle atrophy and metabolic disturbance, which is one of the prominent features during inflammation and infection and characterised by muscle strength loss, protein degradation and carbohydrate metabolism impairment(Reference Crossland, Constantin-Teodosiu and Gardiner1).
During inflammation and infection, pattern recognition receptors such as toll-like receptors (TLR) and nucleotide-binding oligomerisation domain proteins (NOD), as critical components of innate immune response, are activated to produce pro-inflammatory cytokines(Reference Adamczak2, Reference Chen, Yu and Li3). These pro-inflammatory cytokines directly induce muscle protein degradation or lead to muscle atrophy through inhibiting muscle protein kinase B (Akt) and activating Forkhead box O (FOXO) transcription factors and further activating FOXO target genes such as E3-ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger (MuRF) 1, which is the main pathway of protein degradation in skeletal muscle(Reference Crossland, Constantin-Teodosiu and Gardiner1). In addition, the activated FOXO impairs carbohydrate oxidation by activating pyruvate dehydrogenase kinase (PDK) 4 and then inhibiting pyruvate dehydrogenase complex (PDC) activity(Reference Mallinson, Constantin-Teodosiu and Sidaway4).
The inflammatory response (inflammation) is part of innate immunity. It occurs when tissues are injured by bacteria, trauma, toxins, heat or any other cause(Reference Sheriff, Herrmann and Voll5). Long-chain n-3 PUFA such as DHA (22 : 6n-3) and EPA (20 : 5n-3) rich in deep-sea fish oil can inhibit inflammatory response and exert beneficial effects on human clinical studies and animal experiments(Reference De Caterina6, Reference Liu, Chen and Odle7). In addition, n-3 PUFA (EPA or DHA) alleviates muscle atrophy(Reference Whitehouse, Smith and Drake8, Reference Khala and Tisdale9), improves muscle protein synthesis in older, healthy young and middle-aged adults(Reference Smith, Atherton and Reeds10, Reference Smith, Atherton and Reeds11) and decreases PDK activity in human skeletal muscle(Reference Turvey, Heigenhauser and Parolin12). So, the general public is recommended to consume fish oil in many nutritional guidelines(Reference Krauss, Eckel and Howard13). However, the use of fish oil on clinic is often limited because of the characteristics of easy oxidation(Reference Choi, Ruktanonchai and Min14). So, alternative sources of n-3 PUFA existing in plants receive attention. Flaxseed oil, a traditional edible oil, is rich in α-linolenic acid (ALA; 18 : 3n-3). ALA is the metabolic precursor of EPA and DHA(Reference Kouba and Mourot15). Recently, emerging literatures have shown that flaxseed oil inclusion in the diet decreased inflammatory response and exerted anti-inflammatory effects(Reference Thies, Miles and Nebe-von-Caron16, Reference Moura-Assis, Afonso and de Oliveira17). However, unlike fish oil, few studies have been conducted to explore the effect of flaxseed oil on muscle atrophy and carbohydrate oxidation impairment.
The weaned piglet model is suitable for human nutrition research, particularly child and adolescent with muscles undergoing rapid growth(Reference Spurlock and Gabler18, Reference Dunshea and Cox19). Lipopolysaccharide (LPS) is often utilised to establish the model of endotoxaemia(Reference Crossland, Constantin-Teodosiu and Gardiner1). Therefore, in the present study, our objective was to investigate whether flaxseed oil had a protective effect on muscle atrophy and carbohydrates oxidation impairment caused by LPS challenge and to elucidate its molecular mechanism(s) in the weaned piglet model.
Methods
Animal and experimental design
The Animal Care and Use Committee of Wuhan Polytechnic University approved the animal use protocol for this research. Twenty-four weaned crossbred castrated barrows (Duroc × Large White × Landrace; 8·91 (sem 0·21) kg; 35 (sem 1) d of age) were randomly assigned to four treatment groups and allowed ad libitum access to water and feed during a 21-d experimental study; each treatment group had six replicated pens, and each pen had one pig, which was based on our previous studies(Reference Liu, Chen and Odle7, Reference Zhu, Wang and Wang20). In the whole experimental period, all pigs were placed individually in pens (1·80 × 1·10 m) and in good health condition. The ambient temperature was maintained at 22–25°C, and the living environment was in accordance with animal welfare guidelines.
The experiment was conducted as a 2 × 2 factorial arrangement of treatments including dietary treatment (5 % maize oil (Xiwang Food Company) v. 5 % flaxseed oil (Yulongxiang Grain and Oil Company)) and LPS (Escherichia coli serotype 055:B5, Sigma Chemical) challenge (saline v. LPS). On day 21, half of the pigs in each dietary treatment were injected intraperitoneally with 100 μg/kg body weight LPS or the same volume of 0·9 % sterile saline solution according to our previous study(Reference Liu, Chen and Odle7). This dose of LPS could cause acute tissue injury in weaned pigs. The ingredient composition of the basal diet and the fatty acid composition of maize oil and flaxseed oil were shown in our previous study(Reference Zhu, Wang and Wang20).
Sample collection
At 4 h after LPS or saline injection, blood samples were harvested into 10-ml heparinised vacuum tubes (Becton Dickinson Vacutainer System) and centrifuged (3500 g for 10 min) to collect plasma. Plasma from each pig was stored at –80°C until further analysis. After blood collection, pigs were slaughtered under anaesthesia with an intravenous injection of pentobarbital sodium (80 mg/kg body weight), and a portion of gastrocnemius muscle and longissimus dorsi (LD) muscle (approximately 20 g, respectively) was removed and frozen in liquid N2 immediately and then stored at −80°C for mRNA and protein expression analysis. Ooi et al. found piglets with muscle fibre atrophy had high levels of MAFbx in both gastrocnemius muscle and LD muscle(Reference Ooi, da Costa and Edgar21). LPS can induce muscle atrophy by up-regulating mRNA expression of MAFbx and MuRF1(Reference Wan, Chen and Yu22–Reference Zhang, Yu and Lin24). Therefore, these muscles were selected to study muscle atrophy(Reference Ooi, da Costa and Edgar21, Reference Drew, Phaneuf and Dirks25). In addition, the time point of 4 h after LPS or saline injection was selected according to published papers(Reference Liu, Chen and Odle26–Reference Andreasen, Kelly and Berg28).
Fatty acids composition in muscles
Total fat was prepared from muscles according to GB/T 14772-2008(29). The composition of fatty acids in muscles was analysed according to the method of Sun et al.(Reference Sun, Kang and Xie30). Briefly, the muscle (about 5 g) was extracted in a Soxhlet extractor with diethyl ether for 6–12 h; then, the extraction was concentrated in water bath at 70–80°C and dried at 103°C for 1 h. Undecanoic acid (C11 : 0, 1 ml, served as the internal standard) and acetyl chloride methanol solution (4 ml) were added to the extracted fat and then placed in an 80°C water for 2 h. After cooling, 5 ml potassium carbonate solution (7 %) was added into the mix and then mixed and centrifuged at 1200 g for 5 min (4°C). The resulting supernatant was put into a DB-23 capillary column (60·0 µm × 250 µm×0·25 µm; Agilent Technoligies) to analyse the fatty acid composition by gas chromatograph (Agilent 6890; Agilent Technoligies).
Glucose, blood urea nitrogen, insulin, cortisol, glucagon, TNF-α, IL-6 and IL-8 concentrations in plasma
Plasma glucose level was determined by the glucose GOD-PAP assay kit (DiaSys Diagnostic Systems GmbH). Plasma blood urea N (BUN) concentrations were measured by a commercial urea assay kit (Nanjing Jiancheng Bioengineering Institute). Plasma insulin, cortisol and glucagon contents were measured using commercially available 125I RIA assay kits (Beijing North Institute of Biological Technology). Plasma TNF-α, IL-6 and IL-8 levels were determined by using a commercially available porcine ELISA assay kit (R&D Systems). All experimental procedures were conducted according to the manufacturer’s recommendations.
Protein and DNA contents in muscles
The muscle samples were homogenised with a tissue homogeniser (PT-3100D; Kinematica) in ice-cold PBS EDTA (0·05 m-Na3PO4, 2·0 m-NaCl, 2 × 10−3m-EDTA, pH 7·4) using a 1:10 (w/v) ratio. Protein concentration of muscle homogenates was determined by a published method(Reference Lowry, Rosebrough and Farr31) using a detergent-compatible protein assay (Bio-Rad Laboratories) and bovine serum albumin as standards. Muscle DNA content was evaluated by a fluorometric assay(Reference Labarca and Paigen32).
Glycogen and lactate contents and pyruvate dehydrogenase complex quantity in muscles
Glycogen and lactate contents in muscles were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute). PDC quantity was analysed using a commercial ELISA kit (Yuanye Biotech Co., Ltd). All experimental procedures were conducted according to the manufacturer’s recommendations.
mRNA abundance analysis by real-time PCR
The genes expression were measured as previously described(Reference Zhu, Wang and Wang20). Briefly, total RNA was isolated by the Trizol reagent (no. 9108, TaKaRa Biotechnology (Dalian) Co., Ltd). RNA quality for each sample was detected by agarose gel electrophoresis, and its concentration was determined by spectrophotometry based on the OD260:OD280 ratio. cDNA was synthesised using PrimeScript® RT reagent kit (no. RR047A, TaKaRa Biotechnology (Dalian) Co., Ltd) according to the manufacture’s instruction. Real-time PCR assay for the target genes was carried out on a ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies) using a SYBR® Premix Ex TaqTM (Tli RNaseH Plus) quantitative PCR kit (no. RR420A, TaKaRa Biotechnology (Dalian) Co., Ltd). The PCR cycling conditions were 95ºC × 30 s, followed by forty cycles of 95ºC × 5 s and 60ºC × 34 s. The forward and reverse primers for the target genes were designed with Primer Premier 6.0 and synthesised by TaKaRa Biotechnology (Table 1), and the mRNA expression relative to a housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase; GAPDH) was calculated according to the 2−△△CT method(Reference Livak and Schmittgen33).
Table 1. Primer sequences used for real-time PCR
TLR, toll-like receptor; LBP, lipopolysaccharide-binding protein; CD14, cluster of differentiation factor 14; MyD88, myeloid differentiation factor 88; IRAK1, IL-1 receptor-associated kinase 1; TRAF6, TNF receptor-associated factor 6; NOD, nucleotide-binding oligomerisation domain protein; RIPK2, receptor-interacting serine/threonine-protein kinase 2; CCL2, CC chemokine ligand 2; COX2, mitogen-inducible cyclo-oxygenase 2; Akt1, protein kinase B1; FOXO, Forkhead box O; MAFbx, muscle atrophy F-box; MuRF1, muscle RING finger 1; PDK4, pyruvate dehydrogenase kinase 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Protein abundance analysis by Western blot
Quantification of protein expression in muscles was performed as previously described(Reference Liu, Chen and Odle7, Reference Liu, Chen and Odle26). Briefly, supernatant proteins were separated on 12 % SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes for immunoblotting. The primary antibodies including total Akt (no. 9272), phosphorylated Akt (p-Akt, Ser473, no. 9271), total FOXO1 (t-FOXO1, no. 9454) and phosphorylated FOXO1 (p-FOXO1, Ser256, no. 9461) and the secondary antibodies had been described in Kang et al.(Reference Kang, Wang and Wu34). In the present study, the same target protein, including total and phosphorylated proteins, was measured on the same gel by using their respective antibodies. The bands were analysed by densitometry using GeneTools software (Syngene), and the abundance of the phosphorylated proteins was normalised to the total protein contents.
Statistical analysis
The experimental data were analysed using the general linear model procedure of Statistical Analysis System appropriate for a 2 × 2 factorial design. The statistical model included the effects of diet (maize oil v. flaxseed oil) and LPS challenge (saline v. LPS) and their interactions. If there was a significant or a trend interaction observed between LPS challenge and diet, post hoc testing was conducted using Duncan’s multiple comparison tests. Data were expressed as means and standard deviations. The statistical significance level for all analyses was set at P ≤ 0·05, and 0·05 < P < 0·10 was considered as trends. A two-way multivariate ANOVA was used to determine the effect size (partial η 2) and the statistical power of the model. There was a statistically significant interaction effect between diet and LPS on the combined dependent variables (F = 3·287, P = 0·036, Wilks’ Λ = 0·217, partial η 2 = 0·783, the power to detect the effect was 0·789).
Results
Fatty acid composition in muscles
During the whole experimental period, there were no adverse events. The fatty acid composition in muscles is shown in Table 2. Flaxseed oil supplementation increased ALA, eicosatrienoic acid (C20 : 3n-3), EPA and total n-3 PUFA proportions in both gastrocnemius muscle and LD muscle (P < 0·05). However, its inclusion decreased linoleic acid (C18 : 2n-6), eicosatrienoic acid (C20 : 3n-6), arachidonic acid (C20 : 4n-6), total n-6 PUFA proportion and n-6:n-3 ratio in both gastrocnemius muscle and LD muscle (P < 0·05).
Table 2. Effects of flaxseed oil or maize oil supplementation on muscle fatty acid composition after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets*
(Mean values and standard deviations, n 6 (one pig per pen))
a,b,cMean values within a row with unlike superscript letters were significantly different (P < 0·05).
* Total n-6 PUFA and total n-3 PUFA corresponded to the sum of all the detected n-6 or n-3 PUFA.
Glucose, blood urea nitrogen, insulin, cortisol, glucagon and proinflammatory cytokine concentrations in plasma
As shown in Table 3, the piglets challenged with LPS had lower plasma glucose and insulin contents and higher plasma BUN, cortisol, glucagon, TNF-α, IL-6 and IL-8 contents compared with the piglets injected with saline (P < 0·05). There was an interaction observed between LPS challenge and diet for plasma cortisol, IL-6 and IL-8 contents (P < 0·05) and a trend for plasma TNF-α content (P < 0·1). Compared with maize oil, flaxseed oil reduced plasma cortisol, IL-6, IL-8 and TNF-α contents in piglets challenged by LPS (P < 0·05); however, flaxseed oil had no effects on these variables in saline-treated piglets. In addition, there was no interaction observed between LPS challenge and diet for plasma glucose, BUN, insulin and glucagon contents. Flaxseed oil decreased BUN and glucagon contents in both saline-treated and LPS-challenged piglets (P < 0·05).
Table 3. Effects of flaxseed oil or maize oil supplementation on plasma glucose, blood urea nitrogen (BUN), insulin, cortisol, glucagon, and proinflammatory cytokine concentrations after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets
(Mean values and standard deviations, n 6 (one pig per pen))
ND, not detectable.
a,b,cMean values within a row with unlike superscript letters were significantly different (P < 0·05).
Protein:DNA ratio in muscles
As shown in Table 4, LPS challenge decreased protein:DNA ratio in gastrocnemius muscle (P < 0·05). There was a trend for diet × LPS interaction for protein:DNA ratio in gastrocnemius muscle (P < 0·1). Compared with maize oil, flaxseed oil inclusion had no effect on this ratio in gastrocnemius muscle in piglets treated with saline, whereas its inclusion elevated this ratio in gastrocnemius muscle in piglets challenged by LPS (P < 0·05).
Table 4. Effects of flaxseed oil or maize oil supplementation on protein/DNA after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets
(Mean values and standard deviations, n 6 (one pig per pen))
a,bMean values within a row with unlike superscript letters were significantly different (P < 0·05).
Glycogen and lactate contents and pyruvate dehydrogenase complex quantity in muscles
As shown in Table 5, LPS challenge increased lactate content in both gastrocnemius muscle and LD muscle (P < 0·05) and PDC quantity in gastrocnemius muscle (P < 0·05) and led to an increasing trend in PDC quantity in LD muscle (P < 0·1). There was an interaction between LPS challenge and diet for glycogen content and PDC quantity in gastrocnemius muscle (P < 0·05) and lactate content in LD muscle (P < 0·05), and there was a trend for diet × LPS interaction for glycogen content in LD muscle (P < 0·1). Compared with maize oil, flaxseed oil increased glycogen content in gastrocnemius muscle in saline-treated piglets (P < 0·05). In addition, flaxseed oil increased lactate content in LD muscle and PDC quantity in gastrocnemius muscle in both saline-treated and LPS-challenged piglets (P < 0·05).
Table 5. Effects of flaxseed oil or maize oil supplementation on muscle metabolites after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets
(Mean values and standard deviations, n 6 (one pig per pen))
PDC, pyruvate dehydrogenase complex.
a,b,cMean values within a row with unlike superscript letters were significantly different (P < 0·05).
mRNA expression of key genes in toll-like receptor 4 and nucleotide-binding oligomerisation domain proteins signalling pathways in muscles
As shown in Table 6, LPS challenge increased the mRNA expression of TLR4, LPS-binding protein (LBP), cluster of differentiation factor 14 (CD14), myeloid differentiation factor 88, NOD2, receptor-interacting serine/threonine-protein kinase (RIPK) 2, NF -κB, IL-1β, IL-6, IL-8, IL-10, CC chemokine ligand 2 (CCL2), CXCL16 and cyclo-oxygenase 2 in both gastrocnemius muscle and LD muscle (P < 0·05). Flaxseed oil supplementation decreased mRNA expression of LBP and CD14 in both gastrocnemius muscle and LD muscle in LPS-challenged piglets (P < 0·05). In addition, in both saline-injected and LPS-challenged piglets, flaxseed oil inclusion reduced mRNA expression of TNF-α receptor-associated factor (TRAF) 6, NOD2 and RIPK2 in gastrocnemius muscle, and myeloid differentiation factor 88, IL-1 receptor-associated kinase 1 (IRAK1), TRAF6, RIPK2, NF-κB and TNF-α in LD muscle, and increased mRNA expression of IL-10 in gastrocnemius muscle and CCL2 in both gastrocnemius muscle and LD muscle (P < 0·05). There was an interaction between LPS and diet for LBP, CD14 and CCL2 in both gastrocnemius muscle and LD muscle, and IL-1β, IL-6 and cyclo-oxygenase 2 mRNA expression in gastrocnemius muscle, and TLR4, NOD2 and IL-6 mRNA expression in LD muscle (P < 0·05). These variables had no difference between maize oil and flaxseed oil treatment in piglets injected with saline; however, they were all lowered after flaxseed oil was supplemented in piglets challenged with LPS (P < 0·05).
Table 6. Effects of flaxseed oil or maize oil supplementation on muscle mRNA expression of toll-like receptor 4 (TLR4) and nucleotide-binding oligomerisation domain proteins (NOD) and their downstream signals after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets*
(Mean values and standard deviations, n 6 (one pig per pen))
LBP, LPS-binding protein; CD14, cluster of differentiation factor 14; MyD88, myeloid differentiation factor 88; IRAK1, IL-1 receptor-associated kinase 1; TRAF6, TNF-α receptor-associated factor 6; RIPK2, receptor-interacting serine/threonine-protein kinase; CCL2, CC chemokine ligand 2; COX2, cyclo-oxygenase 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
a,b,cMean values within a row with unlike superscript letters were significantly different (P < 0·05).
* All the data were obtained using RT-PCR. GAPDH was the endogenous reference gene, and the pigs fed the maize oil diet and injected with saline were the calibrator sample.
mRNA expression of protein kinase B1/Forkhead box O signalling and their target genes in muscles
As shown in Table 7, there was no interaction observed between LPS challenge and diet for Akt1/FOXO signalling and their target genes. LPS challenge increased the mRNA expression of FOXO1, MuRF1 and PDK4 in both gastrocnemius muscle and LD muscle (P < 0·05); however, these variables were all reduced in both gastrocnemius muscle and LD muscle when flaxseed oil was supplemented (P < 0·05).
Table 7. Effects of flaxseed oil or maize oil supplementation on mRNA expression of protein kinase B1 (Akt1)/Forkhead box O (FOXO) signalling pathways and its target genes after 4 h Escherichia coli lipopolysaccharide (LPS) challenge in weaned piglets*
(Mean values and standard deviations, n 6 (one pig per pen))
MAFbx, muscle atrophy F-box; MuRF1, muscle RING finger 1; PDK4, pyruvate dehydrogenase kinase 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
* All the data were obtained using RT-PCR. GAPDH was the endogenous reference gene, and the pigs fed the maize oil diet and injected with saline were the calibrator sample.
Protein phosphorylation of protein kinase B1 and Forkhead box O1 in muscles
As shown in Fig. 1, there were no interactions between LPS challenge and diet for the p-Akt1:total Akt1 (Fig. 1(a)) and p-FOXO1:t-FOXO1 (Fig. 1(b)). Flaxseed supplementation increased the ratios of p-Akt1:total Akt1 and p-FOXO1:t-FOXO1 in gastrocnemius and LD muscles in both saline-treated and LPS-challenged piglets (P < 0·05).
Fig. 1. Effects of flaxseed oil or maize oil supplementation on the ratios of phosphorylated protein kinase B1(p-Akt1):total Akt1(t-Akt1) (a) and phosphorylated Forkhead box O (p-FOXO1):total FOXO1 (t-FOXO1) (b) in gastrocnemius muscle and longissimus dorsi muscle in weaned piglets after Escherichia coli lipopolysaccharide (LPS) challenge. Values are means (n 6; one piglet per pen), with their standard errors represented by vertical bars. (
) CO-S, piglets fed a maize oil diet and injected with sterile saline; (
) FL-S, piglets fed a flaxseed oil diet and injected with sterile saline; (
) CO-L, piglets fed a maize oil diet and injected with LPS; (
) FL-L, piglets fed a flaxseed oil diet and injected with LPS. Piglets feed flaxseed oil had higher ratios of p-Akt1:t-Akt1 and p-FOXO1:t-FOXO1 in gastrocnemius and longissimus dorsi muscles in both saline-treated and LPS-challenged piglets (P < 0·05).
Discussion
Our previous study had found that fish oil (rich in EPA and DHA) attenuated muscle protein loss induced by LPS challenge in weaned piglets(Reference Liu, Chen and Odle7). Turvey et al. found diet rich in n-3 fatty acids decreased PDK activity in human skeletal muscle(Reference Turvey, Heigenhauser and Parolin12). Flaxseed oil, as a traditional edible oil, is rich in ALA, which is the metabolic precursor of EPA and DHA(Reference Kouba and Mourot15). Therefore, we speculated that flaxseed oil not only had the same function in muscle protein loss as fish oil but also played an important role in attenuating carbohydrate oxidation impairment. In our study, flaxseed oil inclusion increased ALA, EPA and total n-6:n-3 PUFA contents in muscles, which was in agreement with the report of Zhu et al.(Reference Zhu, Wang and Wang20). In addition, Duan et al. reported the optimal n-6:n-3 PUFA ratios of 1:1 and 5:1 exerted beneficial effects on inflammatory system(Reference Duan, Li and Li35). In the present study, we found that flaxseed oil supplementation could optimise this ratio compared with maize oil.
Muscle atrophy is generally associated with excessive loss of muscle protein(Reference Steiner and Lang36). Plasma BUN concentration is an indirect index to reflect muscle protein degradation(Reference Whang and Easter37) and may be useful as an indicator of protein status to quantify N utilisation and excretion rates(Reference Kohn, Dinneen and Russek-Cohen38). In our study, flaxseed oil inclusion decreased BUN content in blood, suggesting that flaxseed oil attenuated LPS-induced muscle atrophy by reducing protein degradation in both normal and stress status. Muscle protein and DNA concentrations are important indexes for muscle mass or muscle protein metabolism(Reference Smith, Atherton and Reeds11). Their ratio can be a sensitive measure for muscle protein mass(Reference Crossland, Constantin-Teodosiu and Gardiner1). In the present study, flaxseed oil inclusion prevented the reduction of protein:DNA ratio after LPS challenge, indicating that flaxseed oil could increase muscle protein mass to prevent muscle atrophy induced by LPS. This result is also consistent with our previous study in fish oil(Reference Liu, Chen and Odle7).
Muscle atrophy has been shown to occur concomitantly with carbohydrate oxidation impairment(Reference Crossland, Constantin-Teodosiu and Gardiner1). Glycogen in skeletal muscle can mainly supply energy for muscle contraction, and its level is associated with exogenous carbohydrate oxidation to energy expenditure(Reference Jeukendrup and Jentjens39). Increased glycogen breakdown indicates an impairment of pyruvate oxidation(Reference Crossland, Constantin-Teodosiu and Gardiner1). In our study, flaxseed oil increased muscle glycogen content in saline-treated piglets, suggesting flaxseed oil inhibited muscle glycogen degradation in the normal status. In addition, the increased lactate accumulation is coincided with the impairment of muscle carbohydrate metabolism(Reference Crossland, Constantin-Teodosiu and Gardiner1). Lactate is mainly produced in anaerobic glycolysis. Alamdari et al. reported that LPS infusion attributed to muscle lactate accumulation because of the reduced PDC activation(Reference Alamdari, Constantin-Teodosiu and Murton40). However, unexpectedly, we found flaxseed oil elevated lactate content in muscles after LPS challenge and we could not explain this result in the present study. PDC mainly controls carbohydrate oxidation by catalysing the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA in skeletal muscle and plays an important role in maintaining metabolic flexibility in skeletal muscle(Reference Park, Jeon and Min41). Moreover, PDC activation can promote immunometabolic homeostasis during sepsis(Reference McCall, Zabalawi and Liu42). PDK4 expression has an inverse relationship with PDC activity(Reference Harris, Bowker-Kinley and Huang43). In the present study, flaxseed oil inclusion reduced PDK4 mRNA expression, indicating that this oil could increase PDC activity, which might result from the increased PDC quantity.
Pro-inflammatory cytokines, both in the circulation and locally at the level of the skeletal muscle, play a critical role in muscle atrophy and carbohydrate metabolism impairment. Previous study reported that ALA could inhibit soluble adhesion molecules release in human umbilical vein endothelial cells challenged by LPS in vitro (Reference Shen, Chen and Xiao44). LBP catalyses the transfer of LPS to CD14. Inhibiting the formation of LPS/LBP/CD14 complexes can protect the host from LPS-induced impairment(Reference Le Roy, Di Padova and Tees45). In the present study, we found flaxseed oil decreased LBP and CD14 mRNA expression in muscles. TLR4 is a critical component of innate immunity(Reference Radin, Sinha and Bhatt46), and it can initiate NF-κB activation through myeloid differentiation factor 88/receptor-associated kinase 1/TRAF6 signalling. Activation of NF-κB then triggers the expression of pro-inflammatory cytokines. In addition, NOD1 and NOD2 also result in NF-κB activation via RIPK2 and stimulates the release of pro-inflammatory cytokines. In our study, consistent with improved muscle protein mass (increased protein:DNA ratio) and carbohydrate metabolism (increased PDC quantity), flaxseed oil decreased plasma TNF-α, IL-6 and IL-8 concentrations and muscle TNF-α, IL-6, IL-1β, and TLR4, NOD2 and their downstream signalling molecules mRNA expression after LPS injection, which was in line with the previous reports in intestine(Reference Zhu, Wang and Wang20). Moreover, as an anti-inflammatory cytokine, IL-10 can prevent TLR-induced inflammatory cytokine production(Reference O’Garra, Barrat and Castro47, Reference Rossato, Curtale and Tamassia48) and down-regulates the expression of IL-1, IL-6, IL-8 and TNF-α (Reference Trifunović, Miller and Debeljak49). CCL2 is essential to mount an adequate inflammatory response to repair acute skeletal muscle injury(Reference Lu, Huang and Ransohoff50). In our study, we found flaxseed oil inclusion could increase IL-10 and CCL2 mRNA expression in muscles. These results indicated that flaxseed oil could attenuate inflammatory response through blocking the formation of LPS/LBP/CD14 complexes, inhibiting TLR4 and NOD signalling pathways and simultaneously stimulating IL-10 and CCL2 mRNA expression, which further alleviate muscle atrophy and carbohydrate oxidation impairment induced by LPS challenge.
Pro-inflammatory cytokines can lead to muscle protein loss directly or via alterations of the Akt/FOXO/ubiquitin-proteasome pathway. Akt/FOXO is a common signalling pathway influencing muscle protein breakdown and synthesis during inflammation(Reference Crossland, Skirrow and Puthucheary51). Phosphorylation and inactivation of FOXO1 induced by Akt1 activation can suppress muscle proteolysis and muscle atrophy by transcriptional inhibition of FOXO target genes such as MAFbx and MuRF1; the latter are the main regulators in protein degradation in skeletal muscle(Reference Milan, Romanello and Pescatore52). Various pathological and physiological conditions can activate these two ubiquitin-ligase enzymes and stimulate muscle proteolysis. In our previous study, we found diet supplemented with fish oil decreased FOXO 1 mRNA abundance and increased phosphorylation of Akt and FOXO1 in muscles. In line with these results, in the present study, we also found flaxseed oil inclusion decreased mRNA expression of FOXO1, MAFbx and MuRF1 and elevated the ratios of p-Akt1:total Akt1 and p-FOXO1:t-FOXO1 in muscles, indicating flaxseed oil could have a positive effect on alleviating muscle protein loss through regulating the Akt/FOXO1/ubiquitin-proteasome pathway signalling pathway.
FOXO signalling has a dual role in both ubiquitin-mediated proteolysis and carbohydrate oxidation impairment. Crossland et al. reported that FOXO could impair carbohydrate oxidation in vivo during sepsis(Reference Crossland, Constantin-Teodosiu and Greenhaff53). Mallinson et al. found increased FOXO activity could up-regulate PDK gene expression, which stimulated PDC phosphorylation and then depressed this complex activity(Reference Mallinson, Constantin-Teodosiu and Sidaway4). PDK4 is a major determinant of PDK activity and is mainly present in muscles(Reference Kwon, Huang and Unterman54, Reference Wu, Sato and Zhao55). In our study, flaxseed oil inclusion reduced PDK4 mRNA expression, which indicates that this oil could improve carbohydrate metabolism to produce more energy for relieving stress induced by LPS.
In addition, fatty acid composition in diet has an effect on the long-term regulation of skeletal muscle PDK(Reference Fryer, Orfali and Holness56). Jucker et al. found safflower oil could reduce glycolytic and oxidative disposal of glucose compared with fish oil(Reference Jucker, Cline and Barucci57). Long-term sucrose-rich diet decreased PDC activity in rats; however, fish oil supplementation could reverse this alteration(Reference Pighin, Karabatas and Rossi58). Stephens et al. found n-6 PUFA partially replaced with n-3 PUFA increased muscle PDC activation(Reference Stephens, Mendis and Shannon59). In our study, flaxseed oil inclusion increased PDC quantity, which might result from the rich contents of n-3 PUFA in flaxseed oil evidenced by the high n-3 PUFA content in muscle.
Previous studies have shown that proinflammatory cytokines can elevate catabolic hormones such as cortisol and glucagon(Reference Keelan60, Reference Ferrannini, Muscelli and Natali61). Cortisol is an inhibitor of muscle protein synthesis through binding to its glucocorticoid receptor, and both glucagon and cortisol can increase protein breakdown and urea formation(Reference Gann, Andre and Roemer62, Reference Møller63). In addition, these hormones can also affect carbohydrate metabolism(Reference Salehzadeh, Al-Khalili and Kulkarni64, Reference Park, Jeon and Go65). Glucocorticoid and glucagon could increase PDK4 mRNA expression(Reference Salehzadeh, Al-Khalili and Kulkarni64, Reference Park, Jeon and Go65). However, glucagon is not generally considered to act in skeletal muscle(Reference Hansen, Abrahamsen and Nishimura66), which indicates glucagon might have an indirect effect on protein metabolism in muscle. In our study, flaxseed oil reduced plasma cortisol content, indicating that flaxseed oil could attenuate protein degradation through decreasing plasma cortisol content after LPS challenge.
As we know, dynamic changes occur in protein metabolism, carbohydrate oxidation, pro-inflammatory cytokine gene expression and protein phosphorylation of signalling molecules in muscles after LPS challenge(Reference Tarabees, Hill and Rauch67–Reference Frost, Nystrom and Lang69). So, measurements taken only at one time point (4 h) are probably not adequate to confirm the roles of signalling molecules and pro-inflammatory mediators in LPS-induced muscle atrophy and carbohydrate oxidation impairment. In addition, besides TLR4 and NOD signalling pathways, many other pathways can induce the production of proinflammatory cytokines. Thus, in future studies, sample will be collected at more time points to investigate the dynamic interplay of flaxseed oil supplementation on muscle atrophy and carbohydrate oxidation. In addition, many other inflammatory pathways should also be investigated.
In summary, diet supplemented with flaxseed oil might have a positive effect on alleviating muscle protein loss and carbohydrates oxidation impairment. These beneficial effects of flaxseed oil on muscles might be associated with regulating the TLR4/NOD and Akt/FOXO signalling pathways. The present study provides a good reference for human nutrition, and flaxseed oil may have a positive role in human health.
Acknowledgements
This research was financially supported by projects of the Wuhan Science and Technology Bureau (2018020401011304), the National Natural Science Foundation of China (31772615), the Project of the Hubei Provincial Department of Education (T201508), the Natural Science Foundation of Hubei Province (2018CFB527) and the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (201804).
The authors’ contributions were as follows: Y. L. and P. K. designed the research; Y. L., P. K., Y. W., X. L., Z. W., X. W., H. Z., C. W., S. Z., H. C. conducted the research. Y. L. and P. K. analysed data and wrote the paper. Y. L. had primary responsibility for the final content.
The authors declare no conflicts of interest.
