Fatty acid metabolism in muscle includes uptake, synthesis and oxidation [24,25,26], but the synthesis is at a slow rate [27]. The main source of fatty acid in muscle tissue includes transport from plasma and hydrolysis from chylomicron and very-low-density-lipoprotein (VLDL) with LPL. Our study found that the concentration of FFA was significantly increased in muscle when pigs were fed betaine, similarly to the studies carried out by Yang et al. [28] and Fernández-Fígares et al. [29]. We speculated that the transport of FFA and/or the hydrolysis may be enhanced. More experiments were carried out regarding factors involved in fatty acid transport in muscle tissue. It is widely recognized that long chain fatty acid (LCFA) cross the plasma membrane via a protein-mediated mechanism. A number of fatty acid transporters have been identified, including fatty acid translocase/cluster of differentiation (FAT/CD36) and fatty acid transport proteins (FATP1) [30]. We found that betaine supplementation up-regulated gene expression for FATP1 and FAT/CD36. Experiments in vitro have shown that over expression of FATP1 increased the uptake of LCFA in cells [31] and studies in vivo documented that muscle-specific over-expression of FAT/CD36 enhanced cellular fatty acid uptake in mice [32]. FABP3, another important protein in fatty acid transportation, plays a role in transporting fatty acid from the sarcolemma to their intracellular sites of metabolism [33]. In muscle cells, the intracellular transport of LCFAs is facilitated to a great extent by FABP3 [34] Additionally, FABP3 is confirmed to be associated with intramuscular fat in pigs [35]. Our studies showed that feeding betaine up-regulated the protein abundance of FABP3. In addition, the gene expression of FABP3 was enhanced when pigs were fed with 2500 mg/kg betaine but no difference was found with 1250 mg/kg-betaine addition. The possible reason maybe that FABP3 expression is translationally rather than transcriptionally regulated [36]. In summary, betaine may promote the uptake of fatty acids in muscle via regulating the expression of FAT/CD36, FATP1 and FABP3. As mentioned above, LPL is the principal enzyme that hydrolyzes circulating triglycerides and it also can increase lipid uptake [37]. The results showed a significant increase in the gene expression of LPL with the addition of 2500 mg/kg betaine, which indicates betaine might enhance lipid uptake as well as chylomicron hydrolysis. The nuclear receptor PPARγ is a central regulator of adipose tissue development and an important modulator of expression in adipocytes [38]. To date, only a limited number of genes are known to be direct targets of PPARγ in adipose tissue. The majority of these encode proteins with direct links to lipid metabolism including LPL, FATP and FAT/CD36 [39, 40]. In present study, the gene expression of PPARγ was significantly higher in betaine-fed groups than the control group. We found that the effect of betaine on PPARγ was similar to its downstream target genes. All these results were similar to Albuquerque [41] and imply that betaine may facilitate fatty acids uptake in muscle via affecting key factors involved in FFA uptake, and the specific regulation mechanism needs more research.

The concentration of FFA in muscle tissue resultes from the balance of transport and oxidation. As a methyl donor, betaine participates in the biosynthesis of carnitine and because of this, betaine may be related to fatty acid β-oxidation. LCFAs are first transformed into acyl CoA, then transferred into mitochondria after combining with carnitine where it is oxidized. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme that controls the step of combination and malony-CoA is an allosteric inhibitor of CPT1 [42]. Whereas the synthesis of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the activity of the ACC is regulated by phosphorylation of AMPK [43]. Hence, AMPK-ACC-CPT1 is an important signaling pathway to regulate fatty acid β-oxidation in mitochondria. Cai et al. [44] found that gestational dietary betaine supplementation down-regulated expression of ACC in neonatal piglets and Pekkinen et al. [11] found betaine supplementation had an impact on carnitine metabolism in high-fat-fed mice. Our experiment didn’t find significant changes in muscle concentrations of malonyl-CoA or carnitine. The different results might be related to the different experiment condition and the mechanism needs to be further investigated. Increased gene expression and protein expression of CPT1 were up-regulated with betaine addition, which implied betaine may enhance fatty acid β-oxidation in muscle tissue. However, others have shown betaine supplementation reduced the activity of CPT1 and mRNA abundance, and further increased IMF in finishing pigs [Duroc × (Seghers × Seghers)] [15]. We speculate that the effect of betaine addition on CPT1 might be influenced by breed and muscle type. In order to get a better understanding, we further analyzed effects of betaine on AMP-activated protein kinase (AMPK) and PPARα, which are both upstream regulatory factors of CPT1. AMPK is a crucial energy sensor for cells, which can promote the catabolism of fatty acids by enhancing their uptake into mitochondria and their consequent breakdown by beta-oxidation [45]. It was reported that activated AMPK in muscle enhances the gene expression of PPARα and CPT1 [46], and CPT1 also seems to be a target of PPARα [47]. In the current experiment, the gene expression of both PPARα and AMPK were higher in betaine-fed groups as well as protein expression of p-AMPK (the activated form of AMPK). Similar to our previous report in rat liver [12], it can be inferred that betaine affected fatty acid oxidation in muscle via activating AMPK and up-regulated PPARα and CPT1 gene expression.

The effect of betaine supplementation on cholesterol metabolism was of interest. The present study showed that betaine supplementation decreased the concentration of serum cholesterol and HDLC and increased cholesterol level in muscle, which was consistent with the studies by Albuquerque et al. [41] and Yang et al. [20]. However, Matthews et al. [48] and Martins et al. [49] reported that betaine supplemented pigs presented higher serum cholesterol. The efficacy of betaine in regulating the concentration of cholesterol in pigs shows variable results and seems to depend on both animal and dietary factors. Although the results were inconsistent, it seems to indicate that betaine might affect cholesterol partitioning or maybe enhances the transport of cholesterol in pigs, and more research is needed to clarify the specific mechanism.