Hongkui Wei

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.

Transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation.

Intramuscular fat (IMF) is an important trait influencing meat quality, and preadipocyte differentiation is a key factor affecting IMF deposition. Here we compared the transcriptome profiles of porcine intramuscular and subcutaneous preadipocytes during differentiation to gain insight into specific molecular and cellular events associated with intramuscular stromal vascular cell (MSVC) differentiation. RNA-Seq was used to screen for differentially expressed genes (DEGs) during the in vitro differentiation of MSVC and subcutaneous stromal vascular cell (ASVC) on days 0, 2 and 4. A total of 985 DEGs were identified during ASVC differentiation and 1469 DEGs during MSVC differentiation. Among these DEGs, 409 genes were specifically expressed during ASVC differentiation, 893 genes were specifically expressed during MSVC differentiation, and 576 DEGs were co-expressed during ASVC and MSVC differentiation. The expression profiles of DEGs during ASVC or MSVC differentiation were determined by cluster analysis based on Short Time-series Expression Miner (STEM). Four significant STEM profiles (profiles 1, 4, 5, and 14) were determined during ASVC differentiation, and four significant STEM profiles (profiles 1, 4, 11, and 14) were determined during MSVC differentiation. Gene ontology (GO) analysis indicated that DEGs related to adipocyte differentiation were identified to be significantly enriched in both adipose and muscle profile 14. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in adipose profile 14 and muscle profiles 11 and 14 (STEM clustered them into one cluster) showed that the PPAR signaling pathway was significantly enriched in these profiles and four signaling pathways were specifically enriched in muscle profiles 11 and 14. Furthermore, analysis of transcription factor binding sites (TFBS) in the gene set revealed two over-represented transcription factors (NR3C4 and NR3C1), which were specifically significantly enriched in the promoter regions of genes within muscle gene expression profiles 11 and 14.

EPA inhibits the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 pathway in C2C12 myotubes in a PPARγ-dependent manner.

The present study was conducted to evaluate the mechanism by which n-3 PUFA regulates the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 (MuRF1) pathway in C2C12 myotubes. After treatment with 150, 300 or 600 μm-α-linolenic acid (ALA) or -EPA for 24 h in C2C12 myotubes, the levels of phosphorylated IκBα (p-IκBα) and total IκBα were measured by Western blot. Compared with the bovine serum albumin (BSA) control, 150 and 300 μm-ALA and -EPA, respectively, did not affect the total IκBα protein level (P>0·05). However, 600 μm-EPA, but not 600 μm-ALA, prevented IκBα phosphorylation and increased the total IκBα levels (P < 0·01). Furthermore, total nuclear protein was isolated and analysed by the electrophoretic mobility shift assay for NF-κB DNA-binding activity after treatment with 600 μm-ALA or -EPA for 24 h. EPA (600 μm), but not ALA (600 μm), decreased the NF-κB DNA-binding activity when compared with BSA (P < 0·01). It was further observed that 600 μm-EPA caused a 3·38-fold reduction in the levels of MuRF1 mRNA expression compared with BSA (P < 0·01). Additionally, 600 μm-EPA resulted in a 2·3-fold induction of PPARγ mRNA expression (P < 0·01). In C2C12 myotubes, PPARγ knockdown by RNA interference significantly decreased PPARγ mRNA and protein expression to approximately 50 and 60% (P < 0·01), respectively. Interestingly, in C2C12 myotubes with PPARγ knockdown, 600 μm-ALA and -EPA did not affect the levels of p-IκBα and total IκBα, NF-κB DNA-binding activity or MuRF1 mRNA expression when compared with BSA (P>0·05). These results revealed that EPA, but not ALA, inhibited the IκBα/NF-κB/MuRF1 pathway in C2C12 myotubes in a PPARγ-dependent manner.

Effects of dietary n-6:n-3 fatty acid ratio and vitamin E on semen quality, fatty acid composition and antioxidant status in boars

The aim of the present study was to evaluate the effects of dietary n-6:n-3 fatty acid (FA) ratio and vitamin E on the semen quality, FA composition and antioxidant status of boars. Forty-eight Landrace boars were randomly distributed in a 3×2 factorial design with three n-6:n-3 FA ratios (14.4, 6.6 and 2.2) by the inclusion of three oil sources (soybean, fish/soybean, fish) and two vitamin E levels (200 and 400mg/kg). During the 8 weeks of treatment, semen parameters were evaluated. Serum, sperm and seminal plasma samples were taken at 0 and 8 weeks to monitor the FA composition and antioxidant status. Results showed that the 6.6 and 2.2 dietary ratios very effectively increased docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (PUFA) and decreased docosapentaenoic acid (DPA) and n-6:n-3 ratio in spermatozoa. The 6.6 dietary ratio contributed to a greater progressive sperm motility (P<0.05) than the 14.4 and 2.2 dietary ratio, and this ratio also enhanced the superoxide dismutase (SOD) and total antioxidant capacity (TAC) (P<0.05) in seminal plasma more significantly than the other two ratios at week 8. Compared with 200mg/kg supplementation of vitamin E, 400mg/kg supplementation of vitamin E increased the progressive sperm motility, SOD of sperm, TAC and SOD of seminal plasma and serum, and decreased sperm malondialdehyde (MDA) (P<0.05). In conclusion, the 6.6 dietary ratio and 400mg/kg vitamin E supplementation improve progressive sperm motility by modifying the sperm FA composition and antioxidant status.

Feeding a DHA-enriched diet increases skeletal muscle protein synthesis in growing pigs: association with increased skeletal muscle insulin action and local mRNA expression of insulin-like growth factor 1

Dietary n-3 PUFA have been demonstrated to promote muscle growth in growing animals. In the present study, fractional protein synthesis rates (FSR) in the skeletal muscle of growing pigs fed a DHA-enriched (DE) diet (DE treatment) or a soyabean oil (SO) diet (SO treatment) were evaluated in the fed and feed-deprived states. Feeding-induced increases in muscle FSR, as well as the activation of the mammalian target of rapamycin and protein kinase B, were higher in the DE treatment as indicated by the positive interaction between diet and feeding. In the fed state, the activation of eIF4E-binding protein 1 in the skeletal muscle of pigs on the DE diet was higher than that in pigs on the SO diet (P<0·05). Feeding the DE diet increased muscle insulin-like growth factor 1 (IGF-1) expression (P<0·05) and insulin action (as demonstrated by increased insulin receptor (IR) phosphorylation, P<0·05), resulting in increased IR substrate 1 activation in the fed state. However, no difference in plasma IGF-1 concentration or hepatic IGF-1 expression between the two treatments was associated. The increased IGF-1 expression in the DE treatment was associated with increased mRNA expression of the signal transducer and activator of transcription 5A and decreased mRNA expression of protein tyrosine phosphatase, non-receptor type 3 in skeletal muscle. Moreover, mRNA expression of protein tyrosine phosphatase, non-receptor type 1 (PTPN1), the activation of PTPN1 and the activation of NF-κB in muscle were significantly lower in the DE treatment (P<0·05). The results of the present study suggest that feeding a DE diet increased feeding-induced muscle protein synthesis in growing pigs, and muscle IGF-1 expression and insulin action were involved in this action.

GPR120 promotes adipogenesis through intracellular calcium and extracellular signal-regulated kinase 1/2 signal pathway

Numerous researches have demonstrated that GPR120 (also called FFAR4) exerts novel functions in insulin resistance and adipogenesis. However, the molecular mechanism of GPR120-mediated adipogenic differentiation is still unclear. This study was aimed to interpret the relevant function mechanism of GPR120 in the differentiation of 3T3-L1 adipocytes. The results showed that GPR120 expression was dramatically increased along with the adipogenic differentiation of 3T3-L1 adipocytes and the adipogenic ability was significantly inhibited in shGPR120-transfected cells. TUG-891, a selective agonist of GPR120, promoted the intracellular triglyceride accumulation in a dose-dependent manner and did not enhance adipogenesis in shGPR120-transfected cells. Markedly, TUG-891 increased the activation of PPARγ in a GPR120-dependent pathway as assessed by luciferase reporter assay. Furthermore, in the adipogenic differentiation process of 3T3-L1 adipocytes, TUG-891 increased the [Ca(2+)]i and phosphorylation level of ERK1/2. Pretreatment with inhibitors of either ERK1/2 (U0126) or [Ca(2+)]i (BAPTA-AM) notably attenuated the GPR120-mediated adipogenesis. These results show that GPR120 promotes adipogenesis by increasing PPARγ expression via [Ca(2+)]i and ERK1/2 signal pathway in 3T3-L1 adipocytes.

Oregano Essential Oil Improves Intestinal Morphology and Expression of Tight Junction Proteins Associated with Modulation of Selected Intestinal Bacteria and Immune Status in a Pig Model

Oregano essential oil (OEO) has long been used to improve the health of animals, particularly the health of intestine, which is generally attributed to its antimicrobial and anti-inflammatory effects. However, how OEO acts in the intestine of pig is still unclear. This study was aimed at elucidating how OEO promotes the intestinal barrier integrity in a pig model. Pigs were fed a control diet alone or one supplemented with 25 mg/kg of OEO for 4 weeks. The OEO-treated pigs showed decreased (P < 0.05) endotoxin level in serum and increased (P < 0.05) villus height and expression of occludin and zonula occludens-1 (ZO-1) in the jejunum. These results demonstrated that the integrity of intestinal barrier was improved by OEO treatment. The OEO-treated pigs had a lower (P < 0.05) population of Escherichia coli in the jejunum, ileum, and colon than the control. This is in accordance with the greater inactivation (P < 0.05) of inflammation, which was reflected by the mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and nuclear factor κB (NF-κB) signaling pathways and expression of inflammatory cytokines in the jejunum. Our results show that OEO promotes intestinal barrier integrity, probably through modulating intestinal bacteria and immune status in pigs.

A carvacrol-thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger RNA levels of tight junction proteins in jejunal mucosa of weaning piglets.

Recent studies indicate that intestinal oxidative stress and microbiota imbalance is involved in weaning-induced intestinal dysfunction in piglets. We have investigated the effect of feeding a carvacrol-thymol blend supplemented diet on intestinal redox status, selected microbial populations and the intestinal barrier in weaning piglets. The piglets (weaned at 21 days of age) were randomly allocated to two groups with six pens per treatment and 10 piglets per pen. At weaning day (21 days of age), six piglets were sacrificed before weaning to serve as the preweaning group. The weaned group was fed with a basal diet, while the weaned-CB group was fed with the basal diet supplemented with 100 mg/kg carvacrol-thymol (1 : 1) blend for 14 days. On day 7 post-weaning, six piglets from each group were sacrificed to determine intestinal redox status, selected microbial populations, messenger RNA (mRNA) transcript levels of proinflammatory cytokines and biomarkers of intestinal barrier function. Weaning resulted in intestinal oxidative stress, indicated by the increased concentration of reactive oxygen species and thiobarbituric acid-reactive substances present in the intestine. Weaning also reduced the population of Lactobacillus genus and increased the populations of Enterococcus genus and Escherichia coli in the jejunum, and increased mRNA levels of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 (IL-6). In addition, decreased mRNA levels of zonula occludens and occludin in the jejunal mucosa and increased plasma diamine oxidase concentrations indicated that weaning induced dysfunction of the intestinal barrier. On day 7 post-weaning, supplementation with the carvacrol-thymol blend restored weaning-induced intestinal oxidative stress. Compared with the weaned group, the weaned-CB group had an increased population of Lactobacillus genus but reduced populations of Enterococcus genus and E. coli in the jejunum and decreased mRNA levels of TNF-α. The results indicated that weaning induced intestinal oxidative stress and dysfunction of the intestinal barrier. Dietary supplementation with 100 mg/kg carvacrol-thymol (1 : 1) decreased the intestinal oxidative stress and influenced selected microbial populations without changing the biomarkers of intestinal barrier in weaning piglets.

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling.

AbstractBackgroundThe directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.ResultsThis study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.ConclusionsTogether, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

Cloning and characterization of spliced variants of the porcine G protein coupled receptor 120.

The polyunsaturated fatty acids (PUFAs) receptor GPR120 exerts a significant impact on systemic nutrient homeostasis in human and rodents. However, the porcine GPR120 (pGPR120) has not been well characterized. In the current study, we found that pGPR120 had 3 spliced variants. Transcript 1 encoded 362-amino acids (aa) wild type pGPR120-WT, which shared 88% homology with human short form GPR120. Transcript 1 was the mainly expressed transcript of pGPR120. It was expressed predominantly in ileum, jejunum, duodenum, spleen, and adipose. Transcript 3 (coding 320-aa isoform) was detected in spleen, while the transcript 2 (coding 310-aa isoform) was only slightly expressed in spleen. A selective agonist for human GPR120 (TUG-891) and PUFAs activated SRE-luc and NFAT-luc reporter in HEK293T cells transfected with construct for pGPR120-WT but not pGPR120-V2. However, 320-aa isoform was not a dominant negative isoform. The extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in cells transfected with construct for pGPR120-WT were well activated by PUFAs, especially n-3 PUFA. These results showed that although pGPR120 had 3 transcripts, transcript 1 which encoded pGPR120-WT was the mainly expressed transcript. TUG-891 and PUFAs, especially n-3 PUFA, well activated pGPR120-WT. The current study contributed to dissecting the molecular regulation mechanisms of n-3 PUFA in pigs.