Dynamic transcriptome profiles of skeletal muscle growth and development in Shaziling and Yorkshire pigs using RNA-sequencing.

BACKGROUND: We previously demonstrated that Shaziling and Yorkshire pigs differ in growth rate and meat quality. However, the molecular mechanisms responsible for such phenotypic differences remain unclear. In the present study, we performed a transcriptomic analysis of 36 longissimus dorsi (LM) and 36 soleus (SM) muscle samples from Shaziling and Yorkshire pigs at six postnatal stages (30, 60, 90, 150, 210 and 300 days) to explore the differences in postnatal skeletal muscle of Shaziling and Yorkshire pigs. RESULTS: Muscle morphological changes and the number of differentially expressed genes indicated the two stages of 60-90 days and 150-210 days were critical for the muscle growth and development in Shaziling pigs. Genes such as FLNC, COL1A1, NRAP, SMYD1, TNNI3, CRYAB and PDLIM3 played vital roles in the muscle growth, and genes such as CCDC71L, LPIN1, CPT1A, UCP3, NR4A3 and PDK4 played dominant roles in the lipid metabolism. Additionally, in contrast to the LM, the percentage of slow-twitch muscle fibers in the SM of both breeds consistently decreased from 30 to 150 days of age, but there was a significant rebound at 210 days of age. However, the percentage of slow-twitch muscle fibers in the SM of Shaziling pigs was higher than that in Yorkshire pigs, which may be associated with the calcium signaling pathway and the PPARß/δ signaling pathway. CONCLUSION: The present study detected two critical periods and many functional genes for the muscle growth and development of Shaziling pigs, and showed differences in muscle fiber characteristics between Shaziling and Yorkshire pigs. © 2024 Society of Chemical Industry.

Biology and Roles in Diseases of Selenoprotein I Characterized by Ethanolamine Phosphotransferase Activity and Antioxidant Potential.

Selenoprotein I (SELENOI) has been demonstrated to be an ethanolamine phosphotransferase (EPT) characterized by a nonselenoenzymatic domain and to be involved in the main synthetic branch of phosphatidylethanolamine (PE) in the endoplasmic reticulum. Therefore, defects of SELENOI may affect the health status through the multiple functions of PE. On the other hand, selenium (Se) is covalently incorporated into SELENOI as selenocysteine (Sec) in its peptide, which forms a Sec-centered domain as in the other members of the selenoprotein family. Unlike other selenoproteins, Sec-containing SELENOI was formed at a later stage of animal evolution, and the high conservation of the structural domain for PE synthesis across a wide range of species suggests the importance of EPT activity in supporting the survival and evolution of organisms. A variety of factors, such as species characteristics (age and sex), diet and nutrition (dietary Se and fat intakes), SELENOI-specific properties (tissue distribution and rank in the selenoproteome), etc., synergistically regulate the expression of SELENOI in a tentatively unclear interaction. The N- and C-terminal domains confer 2 distinct biochemical functions to SELENOI, namely PE regulation and antioxidant potential, which may allow it to be involved in numerous physiological processes, including neurological diseases (especially hereditary spastic paraplegia), T cell activation, tumorigenesis, and adipocyte differentiation. In this review, we summarize advances in the biology and roles of SELENOI, shedding light on the precise regulation of SELENOI expression and PE homeostasis by dietary Se intake and pharmaceutical or transgenic approaches to modulate the corresponding pathological status.

Fecal miR-142a-3p from dextran sulfate sodium-challenge recovered mice prevents colitis by promoting the growth of Lactobacillus reuteri.

Feces are enriched with microRNAs (miRNAs) that shape the gut microbiota. These miRNAs are differentially expressed in the feces of healthy and diseased subjects. However, whether fecal miRNAs in subjects with inflammatory bowel diseases are involved in regulating microbiota composition and whether they have any beneficial effects remains unknown. Here, we studied the fecal microbiome composition and miRNA abundance in mice with dextran sulfate sodium (DSS)-induced colitis and mice at the recovery phase to explore different miRNAs expressed, their relations with microbial abundance, and their effects on colitis. We found that miR-142a-3p expression was significantly increased in the feces of mice recovered from colitis and that it could alleviate disease symptoms in mice treated with DSS in a microbiome-dependent manner. Specifically, miR-142a-3p promoted the growth of Lactobacillus reuteri, which had a high abundance in the feces of mice recovered from colitis, by regulating transcripts of polA and locus tag LREU_RS03575. Moreover, L. reuteri, as well as its metabolite reuterin, could alleviate DSS-induced disease symptoms. These results highlight the role of fecal miR-142a-3p in the prevention of colitis. We propose that the feces of subjects who have recovered from diseases might be enriched with miRNAs with preventive effects against those diseases.

Selenium-Enriched Cardamine violifolia Increases Selenium and Decreases Cholesterol Concentrations in Liver and Pectoral Muscle of Broilers.

BACKGROUND: Supernutrition of selenium (Se) in an effort to produce Se-enriched meat may inadvertently cause lipid accumulation. Se-enriched Cardamine violifolia (SeCv) contains >80% of Se in organic forms. OBJECTIVES: This study was to determine whether feeding chickens a high dose of SeCv could produce Se-biofortified muscle without altering their lipid metabolism. METHODS: Day-old male broilers were allocated to 4 groups (6 cages/group and 6 chicks/cage) and were fed either a corn-soy base diet (BD, 0.13-0.15 mg Se/kg), the BD plus 0.5 mg Se/kg as sodium selenite (SeNa) or as SeCv, or the BD plus a low-Se Cardamine violifolia (Cv, 0.20-0.21mg Se/kg). At week 6, concentrations of Se and lipid and expression of selenoprotein and lipid metabolism-related genes were determined in the pectoral muscle and liver. RESULTS: The 4 diets showed no effects on growth performance of broilers. Compared with the other 3 diets, SeCv elevated (P < 0.05) Se concentrations in the pectoral muscle and liver by 14.4-127% and decreased (P < 0.05) total cholesterol concentrations by 12.5-46.7% and/or triglyceride concentrations by 28.8-31.1% in the pectoral muscle and/or liver, respectively. Meanwhile, SeCv enhanced (P < 0.05) muscular α-linolenic acid (80.0%) and hepatic arachidonic acid (58.3%) concentrations compared with SeNa and BD, respectively. SeCv downregulated (P < 0.05) the cholesterol and triglyceride synthesis-related proteins (sterol regulatory element binding transcription factor 2 and diacylglycerol O-acyltransferase 2) and upregulated (P < 0.05) hydrolysis and ß-oxidation of fatty acid-related proteins (lipoprotein lipase, fatty acid binding protein 1, and carnitine palmitoyltransferase 1A), as well as selenoprotein P1 and thioredoxin reductase activity in the pectoral muscle and/or liver compared with SeNa. CONCLUSIONS: Compared with SeNa, SeCv effectively raised Se and reduced lipids in the liver and muscle of broilers. The effect was mediated through the regulation of the cholesterol and triglyceride biosynthesis and utilization-related genes.

Advanced single-cell pooled CRISPR screening identifies C19orf53 required for cell proliferation based on mTORC1 regulators.

Multiplexed single-cell CRISPR screening has accelerated the systematic dissection of biological discoveries; however, the efficiency of CRISPR-based gene knockout has inherent limitations. Here, we present DoNick-seq, an advanced method for facilitating gene knockout and reducing off-target activity. We re-engineered two popular plasmid constructs suitable for use in pooled CRISPR screening of the single-cell transcriptome. We then used DoNick-seq to probe mTORC1 regulators and obtain genomic perturbation and transcriptome profiles from the same cell. Thus, DoNick-seq enabled us to simultaneously evaluate multiple gene interactions and the effect of amino acid depletion. By analyzing more than 20,000 cells from two cell lines, DoNick-seq efficiently identified gene targets, cell numbers, and cellular profiles. Our data also revealed the characteristics of mTORC1 negative and positive regulators, thereby shedding new insights into the mechanisms regulating cell growth and inhibition. We demonstrate that mTORC1 hyperactivation exhausts cellular free amino acids via increased proliferation ability. Furthermore, DoNick-seq identified the gene C19orf53, which mediates excessive cell proliferation, resulting in metabolic imbalance, and greatly enhances oxidative stress in response to toxins. Thus, our findings suggest that DoNick-seq facilitates high-throughput functional dissection of complex cellular responses at the single-cell level and increases the accuracy of CRISPR single-cell transcriptomics.

Mitochondrial Iron Metabolism: The Crucial Actors in Diseases.

Iron is a trace element necessary for cell growth, development, and cellular homeostasis, but insufficient or excessive level of iron is toxic. Intracellularly, sufficient amounts of iron are required for mitochondria (the center of iron utilization) to maintain their normal physiologic function. Iron deficiency impairs mitochondrial metabolism and respiratory activity, while mitochondrial iron overload promotes ROS production during mitochondrial electron transport, thus promoting potential disease development. This review provides an overview of iron homeostasis, mitochondrial iron metabolism, and how mitochondrial iron imbalances-induced mitochondrial dysfunction contribute to diseases.

Balanced branched-chain amino acids modulate meat quality by adjusting muscle fiber type conversion and intramuscular fat deposition in finishing pigs.

BACKGROUND: Pork is an important food for humans and improving the quality of pork is closely related to human health. This study was designed to investigate the effects of balanced branched-chain amino acid (BCAA)-supplemented protein-restricted diets on meat quality, muscle fiber types, and intramuscular fat (IMF) in finishing pigs. RESULTS: The results showed that, compared with the normal protein diet (160 g kg-1 crude protein), the reduced-protein diet (120 g kg-1 crude protein) supplemented with BCAAs to the ratio of 2:1:2 not only had higher average daily gain (P < 0.05) and carcass weight (P < 0.05) but also improved meat tenderness and juiciness by decreasing shear force (P < 0.05) and increasing water-holding capacity (P < 0.05). In particular, this treatment showed higher (P < 0.05) levels of phospho-acetyl-CoA carboxylase (P-ACC) and peroxisome proliferation-activated receptor-γ (PPARγ), and lower (P < 0.05) levels of P-adenosine 5'-monophosphate (AMP)-activated protein kinase (P-AMPK), increasing the composition of IMF and MyHC I (P < 0.05) in the longissimus dorsi muscle (LDM). In terms of health, this group increased eicosapentaenoic acid (EPA) (P < 0.01) and desirable hypocholesterolemic fatty acids (DHFA) (P < 0.05), and decreased atherogenicity (AI) (P < 0.01) and hypercholesterolemic saturated fatty acids (HSFA) (P < 0.05). CONCLUSION: Our findings suggest a novel role for a balanced BCAA-supplemented restricted protein (RP) diet in the epigenetic regulation of more tender and healthier pork by increasing IMF deposition and fiber type conversion, providing a cross-regulatory molecular basis for revealing the nutritional regulation network of meat quality. © 2021 Society of Chemical Industry.

Dietary addition of fermented sorghum distiller's dried grains with soluble improves carcass traits and meat quality in growing-finishing pigs.

The fermented sorghum distiller's dried grain with soluble (FS-DDGS) contains numerous nutrients, yet its nutritional effects on growing-finishing pigs remain unclear. The present study evaluated the effects of dietary FS-DDGS addition on growth performance, carcass traits, and meat quality in growing-finishing pigs. A total of 48 healthy male crossbred (Large White × Landrace × Duroc) barrows with initial body weight (BW) of 39.95 ± 2.15 kg were allocated to one of four dietary treatments (12 pigs per treatment). The dietary treatments were as follows: basal diet without (FS-DDGS0 group) or with 50 g/kg (FS-DDGS50 group), 100 g/kg (FS-DDGS100 group), or 150 g/kg (FS-DDGS150 group) FS-DDGS, respectively. Results showed that there were no significant differences in the final BW, average daily gain, average daily feed intake, and feed to gain ratio among these four groups. However, dietary FS-DDGS addition increased (linear, P < 0.05) the pH24h value, contents of ash, crude protein, and proline in Longissimus dorsi muscle, and alanine, arginine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and tyrosine in Biceps femoris (BF) muscle, when compared with the control group. In addition, dietary FS-DDGS addition decreased (linear, P < 0.05) the drip loss, yellowness (b*) value, and lightness (L*) value, while quadratically improved (P < 0.05) the total bone percentage and glycine and proline contents in BF muscle compared with the control group. Collectively, these findings suggested that dietary FS-DDGS addition could improve the carcass traits and meat quality in growing-finishing pigs although further research is needed to explore the underlying mechanisms.

Gut microbiota mediates the protective effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) against obesity induced by high-fat diets.

Obesity increases the risk of developing insulin resistance and diabetes and is a major public health concern. Our previous study shows that dietary ß-hydroxy-ß-methylbutyrate (HMB) improves lipid metabolism in a pig model. However, it remains unclear whether HMB blocks obesity through gut microbiota. In this study, we found that HMB reduced body weight, alleviated the whitening of brown adipose tissue, and improved insulin resistance in mice fed a high-fat diet (HFD). High-throughput pyrosequencing of the 16S rRNA demonstrated that HMB administration significantly reversed the gut microbiota dysbiosis in HFD-fed mice, including the diversity of gut microbiota and relative abundances of Bacteroidetes and Firmicutes. Moreover, microbiota transplantation from HMB-treated mice attenuated HFD-induced lipid metabolic disorders. Furthermore, HFD-fed mice showed lower short-chain fatty acids, whereas administration of HMB increased the propionic acid production. Correlation analysis identified a significant correlation between propionic acid production and the relative Bacteroidetes abundance. Sodium propionate treatment also attenuated HFD-induced lipid metabolic disorders. Collectively, our results indicated that HMB might be used as a probiotic agent to reverse HFD-induced obesity, and the potential mechanism was associated with reprogramming gut microbiota and metabolism, especially Bacteroidetes-mediated propionic acid production. In future studies, more efforts should be made to confirm and expand the beneficial effects of HMB to human models.-Duan, Y., Zhong, Y., Xiao, H., Zheng, C., Song, B., Wang, W., Guo, Q., Li, Y., Han, H., Gao, J., Xu, K., Li, T., Yin, Y., Li, F., Yin, J., Kong, X. Gut microbiota mediates the protective effects of dietary ß-hydroxy-ß-methylbutyrate (HMB) against obesity induced by high-fat diets.

α-Ketoisocaproate and ß-hydroxy-ß-methyl butyrate regulate fatty acid composition and lipid metabolism in skeletal muscle of growing pigs.

OBJECTIVES: This study aims to investigate the effects and roles of excess leucine (Leu) versus its metabolites α-ketoisocaproate (KIC) and ß-hydroxy-ß-methyl butyrate (HMB) on fatty acid composition and lipid metabolism in skeletal muscle of growing pigs. METHODS AND RESULTS: Thirty-two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed one of the four diets (basal diet, L-Leu, KIC-Ca and HMB-Ca) for 45 days. Results indicated that dietary treatments did not affect the intramuscular fat (IMF) content (p > 0.05), but differently influenced the fatty acid composition of longissimus dorsi muscle (LM) and soleus muscle (SM). In particular, the proportion of N3 PUFA specifically in LM was significantly decreased in the Leu group and increased in both KIC and HMB group relative to the basal diet group (p < 0.05). Furthermore, pigs fed KIC-supplemented diets exhibited decreased expression of FATP-1, ACC, ATGL, C/EBPα, PPARγ and SREBP-1c in LM and increased expression of FATP-1, FAT/CD36, ATGL and M-CPT-1 in SM relative to the basal diet control (p < 0.05). CONCLUSIONS: These findings indicated that doubling dietary Leu content decreased the percentage of N3 PUFA mainly in glycolytic skeletal muscle, whereas KIC and HMB improved muscular fatty acid composition and altered lipid metabolism in skeletal muscle of growing pigs. The mechanism of action of KIC might be related to the TFs, and the mechanism of action of HMB might be associated with the AMPK-mTOR signalling pathway.

Effects of dietary ramie powder at various levels on growth performance, antioxidative capacity and fatty acid profile of finishing pigs.

This study was conducted to evaluate the effects of ramie (Boehmeria nivea, previously known as a fibre crop and also called "China grass") included in diets on growth performance, antioxidative capacity and muscular fatty acid profile of finishing pigs. A total of 180 Xiangcun Black pigs (initial body weight =70.71 ± 1.21 kg) were randomly allotted to 1 of 5 dietary treatments with six pens of six pigs per pen. The pigs were provided a basal diet or a diet contained 3%, 6%, 9% or 12% of ramie powder during a 50-day experiment period. The results showed that the inclusion of ramie increased (quadratic, p < 0.05) the average daily gain (ADG) and gain:feed ratio (G:F) with the highest value of ADG and G:F in 3% ramie group, but ramie content in the diet up to 9% reduced the growth performance of the pigs compared with that of 3% ramie group. The activity of serum total superoxide dismutase (SOD) was increased (linear, p < 0.05) by ramie, while content of malondialdehyde was decreased (linear, p < 0.05). As increasing the dietary ramie level, the mRNA expression level of SOD1 was increased quadratically (p < 0.05) in muscle tissues. Moreover, the addition of ramie linearly increased (p < 0.05) polyunsaturated fatty acids content, whereas it linearly reduced (p < 0.05) the lipid indices of atherogenicity (AI) and thrombogenicity (TI) in muscle tissues, and lower values of AI and TI reflect a "healthier" fat composition. The results indicated that ramie in a diet not more than 9% may improve antioxidative capacity with no detrimental impact on growth performance of Chinese native finishing pigs; meanwhile, it could beneficially change the pork fatty acid pattern which has a positive impact on consumer's health.

Dietary mulberry leaf powder affects growth performance, carcass traits and meat quality in finishing pigs.

This study was conducted to evaluate the effect of mulberry leaves as an alternative source of protein on growth performance, carcass traits and meat quality in finishing pigs. A total of 180 Xiangcun Black pigs were randomly assigned to five treatment groups with six pens of six pigs per pen. The pigs were provided with a basal diet or a diet contained 3%, 6%, 9% or 12% of mulberry leaf powder during a 50-day experiment period. The results showed that dietary mulberry leaf powder had no negative effect on growth performance in Xiangcun Black pigs, except in the 12% mulberry group, where final body weight and average daily gain decreased (p < .05) and feed to gain ratio of the pigs increased (p < .05). Dietary mulberry inclusion decreased (quadratic, p < .05) the back fat thickness, fibre mean cross-sectional area (CSA) in the longissimus dorsi (LD) muscle and mRNA expression levels of myosin heavy chain (MyHC) IIb in LD and biceps femoris (BF) muscles, while increased (linear or quadratic, p < .05) the plasma concentration of albumin, levels of crude protein (CP), inosine monophosphate (IMP) and several amino acids in muscle tissues. When compared with the other groups, the 9% mulberry diet increased (p < .05) loin-eye area and contents of CP and IMP in muscles, while decreased (p < .05) plasma activity of cholinesterase and concentrations of uric acid and urea. The 6% mulberry diet had the lowest fibre mean CSA and shear force and increased total fibre number of the LD muscle, when compared with the other groups. These results suggest that including mulberry in the diet at <12% is an effective feed crop to improve meat quality and the chemical composition of muscle without negatively affecting growth performance.

Leucine alone or in combination with glutamic acid, but not with arginine, increases biceps femoris muscle and alters muscle AA transport and concentrations in fattening pigs.

Forty-eight Duroc × Large White × Landrace pigs with an average initial body weight of 77.09 ± 1.37 kg were used to investigate the effects of combination of leucine (Leu) with arginine (Arg) or glutamic acid (Glu) on muscle growth, free amino acid profiles, expression levels of amino acid transporters and growth-related genes in skeletal muscle. The animals were randomly assigned to one of the four treatment groups (12 pigs/group, castrated male:female = 1:1). The pigs in the control group were fed a basal diet (13% Crude Protein), and those in the experimental groups were fed the basal diet supplemented with 1.00% Leu (L group), 1.00% Leu + 1.00% Arg (LA group) or 1.00% Leu + 1.00% Glu (LG group). The experiment lasted for 60 days. Results showed an increase (p < 0.05) in biceps femoris (BF) muscle weight in the L group and LG group relative to the basal diet group. In longissimus dorsi (LD) muscle, Lys, taurine and total essential amino acid concentration increased in the LG group relative to the basal diet group (p < 0.05). In LG group, Glu and carnosine concentrations increased (p < 0.05) in the BF muscle, when compared to the basal diet group. The Leu and Lys concentrations of BF muscle were lower in the LA group than that in the L group (p < 0.05). A positive association was found between BF muscle weight and Leu concentration (p < 0.05). The LG group presented higher (p < 0.05) mRNA levels of ASCT2, LAT1, PAT2, SANT2 and TAT1 in LD muscle than those in the basal diet group. The mRNA levels of PAT2 and MyoD in BF muscle were upregulated (p < 0.05) in the LG group, compared with those in the basal diet group. In conclusion, Leu alone or in combination with Glu is benefit for biceps femoris muscle growth in fattening pig.

ß-Hydroxy-ß-methylbutyrate, mitochondrial biogenesis, and skeletal muscle health.

The metabolic roles of mitochondria go far beyond serving exclusively as the major producer of ATP in tissues and cells. Evidence has shown that mitochondria may function as a key regulator of skeletal muscle fiber types and overall well-being. Maintaining skeletal muscle mitochondrial content and function is important for sustaining health throughout the lifespan. Of great importance, ß-hydroxy-ß-methylbutyrate (HMB, a metabolite of L-leucine) has been proposed to enhance the protein deposition and efficiency of mitochondrial biogenesis in skeletal muscle, as well as muscle strength in both exercise and clinical settings. Specifically, dietary supplementation with HMB increases the gene expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), which represents an upstream inducer of genes of mitochondrial metabolism, coordinates the expression of both nuclear- and mitochondrion-encoded genes in mitochondrial biogenesis. Additionally, PGC-1α plays a key role in the transformation of skeletal muscle fiber type, leading to a shift toward type I muscle fibers that are rich in mitochondria and have a high capacity for oxidative metabolism. As a nitrogen-free metabolite, HMB holds great promise to improve skeletal muscle mass and function, as well as whole-body health and well-being of animals and humans.

Effects of supplementation with branched-chain amino acids to low-protein diets on expression of genes related to lipid metabolism in skeletal muscle of growing pigs.

Branched-chain amino acids (BCAA), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in energy homeostasis and lipid metabolism in addition to their other functions, such as in protein metabolism. This study investigated the effects of different dietary BCAA ratios on the intramuscular fat (IMF) content and fatty acid composition in different location of skeletal muscles, including the longissimus dorsi (LD), biceps femoris (BF), and psoas major (PM) muscles of growing pigs, and also examined the mRNA expression levels of genes involved in lipid metabolism in these muscle tissues. The experiment was performed on 40 growing pigs (Large White × Landrace) with a similar initial weight (9.85 ± 0.35 kg). The pigs were randomly assigned to one of five diets: diet A was a positive control and contained 20 % crude protein (CP) with a Leu:Ile:Val ratio of 1:0.51:0.63 according to the recommendation of the National Research Council (NRC); for diets B to E, the CP level was reduced to 17 %, and the Leu:Ile:Val ratios were 1:1:1, 1:0.75:0.75, 1:0.51:0.63, and 1:0.25:0.25, respectively. No significant difference was observed in the average feed intake and feed efficiency of the pigs fed the low protein diet (17 % CP) with BCAA treatments relative to the positive control. However, there was a tendency for increased feed efficiency of the 1:0.75:0.75 group compared with the 1:1:1 group (P = 0.09). The BCAA ratio of 1:0.75:0.75 (17 % CP) increased the IMF content of BF muscle (P < 0.01). Moreover, varied dietary BCAA supplementation with a reduced protein level had different effects on the fatty acid composition of the LD, BF, and PM muscles. The BCAA ratio of 1:0.51:0.63-1:0.75:0.75 (17 % CP) significantly lowered the ratio of n-6 to n-3 polyunsaturated fatty acid in these muscles compared with the positive control group (20 % CP). This effect was associated with an increase in mRNA expression levels of acetyl-CoA carboxylase, lipoprotein lipase, fatty acid transport protein, and fatty acid binding protein 4 in the muscles (P < 0.05). The results indicated that the reduced protein diet (17 % CP) with the BCAA ratio within 1:0.25:0.25-1:0.75:0.75 could increase the IMF content in BF muscle and significantly improve the fatty acid composition in different skeletal muscles accompanied by changes in the expression of genes involved in lipid metabolism, compared with those in the pigs that received adequate dietary protein (20 %), which might result in improved eating quality and nutritional value of the meat.

The role of leucine and its metabolites in protein and energy metabolism.

Leucine (Leu) is a nutritionally essential branched-chain amino acid (BCAA) in animal nutrition. It is usually one of the most abundant amino acids in high-quality protein foods. Leu increases protein synthesis through activation of the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle, adipose tissue and placental cells. Leu promotes energy metabolism (glucose uptake, mitochondrial biogenesis, and fatty acid oxidation) to provide energy for protein synthesis, while inhibiting protein degradation. Approximately 80 % of Leu is normally used for protein synthesis, while the remainder is converted to α-ketoisocaproate (α-KIC) and ß-hydroxy-ß-methylbutyrate (HMB) in skeletal muscle. Therefore, it has been hypothesized that some of the functions of Leu are modulated by its metabolites. Both α-KIC and HMB have recently received considerable attention as nutritional supplements used to increase protein synthesis, inhibit protein degradation, and regulate energy homeostasis in a variety of in vitro and in vivo models. Leu and its metabolites hold great promise to enhance the growth and health of animals (including humans, birds and fish).

Recent Advances in Understanding Amino Acid Sensing Mechanisms that Regulate mTORC1.

The mammalian target of rapamycin (mTOR) is the central regulator of mammalian cell growth, and is essential for the formation of two structurally and functionally distinct complexes: mTORC1 and mTORC2. mTORC1 can sense multiple cues such as nutrients, energy status, growth factors and hormones to control cell growth and proliferation, angiogenesis, autophagy, and metabolism. As one of the key environmental stimuli, amino acids (AAs), especially leucine, glutamine and arginine, play a crucial role in mTORC1 activation, but where and how AAs are sensed and signal to mTORC1 are not fully understood. Classically, AAs activate mTORC1 by Rag GTPases which recruit mTORC1 to lysosomes, where AA signaling initiates. Plasma membrane transceptor L amino acid transporter 1 (LAT1)-4F2hc has dual transporter-receptor function that can sense extracellular AA availability upstream of mTORC1. The lysosomal AA sensors (PAT1 and SLC38A9) and cytoplasmic AA sensors (LRS, Sestrin2 and CASTOR1) also participate in regulating mTORC1 activation. Importantly, AAs can be sensed by plasma membrane receptors, like G protein-coupled receptor (GPCR) T1R1/T1R3, and regulate mTORC1 without being transported into the cells. Furthermore, AA-dependent mTORC1 activation also initiates within Golgi, which is regulated by Golgi-localized AA transporter PAT4. This review provides an overview of the research progress of the AA sensing mechanisms that regulate mTORC1 activity.

Key mediators of intracellular amino acids signaling to mTORC1 activation.

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

Methionine deficiency reduces autophagy and accelerates death in intestinal epithelial cells infected with enterotoxigenic Escherichia coli.

Infections by enterotoxigenic Escherichia coli (ETEC) result in large economic losses to the swine industry worldwide. Dietary supplementation with amino acids has been considered as a potential mechanism to improve host defenses against infection. The goal of this study was to determine whether methionine deprivation alters ETEC interactions with porcine intestinal epithelial cells. IPEC-1 cells were cultured in media with or without L-methionine. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. Methionine deprivation inhibited IPEC-1 cell autophagic responses, suggesting that the increased cytotoxicity of ETEC to methionine-deprived IPEC-1 cells might be due to defects in autophagy.

Effects of dietary n-6:n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs.

Revealing the expression patterns of fatty acid and amino acid transporters as affected by dietary n-6:n-3 PUFA ratio would be useful for further clarifying the importance of the balance between n-6 and n-3 PUFA. A total of ninety-six finishing pigs were fed one of four diets with the ratio of 1:1, 2·5:1, 5:1 and 10:1. Pigs fed the dietary n-6:n-3 PUFA ratio of 5:1 had the highest (P< 0·05) daily weight gain, and those fed the dietary n-6:n-3 PUFA ratio of 1:1 had the largest loin muscle area (P< 0·01). The concentration of n-3 PUFA was raised as the ratio declined (P< 0·05) in the longissimus dorsi and subcutaneous adipose tissue. The contents of tryptophan, tasty amino acids and branched-chain amino acids in the longissimus dorsi were enhanced in pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1. The mRNA expression level of the fatty acid transporter fatty acid transport protein-1 (FATP-1) was declined (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1, and increased (P< 0·05) in the subcutaneous adipose tissue of pigs fed the dietary n-6:n-3 PUFA ratios of 5:1 and 10:1. The expression profile of FATP-4 was similar to those of FATP-1 in the adipose tissue. The mRNA expression level of the amino acid transceptors LAT1 and SNAT2 was up-regulated (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1 and 2·5:1. In conclusion, maintaining the dietary n-6:n-3 PUFA ratios of 1:1-5:1 would facilitate the absorption and utilisation of fatty acids and free amino acids, and result in improved muscle and adipose composition.