The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated CC myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by small interfering (si)RNA-mediated AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca/calmodulin-dependent protein kinase (CaMKII)δ knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13, or Rab14 reduced, in part, GLUT4 translocation induced by EPS, whereas only Rab8a, or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13, and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

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Both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, but the synergistic action of these mechanical cues on the biological behaviors of mesenchymal stem cells (MSCs) is still not well-known. In the present work, a homemade oscillatory flow device was applied to investigate the effects of matrix stiffness on MSCs survival, distribution, and osteogenic differentiation in three-dimensional (3D) conditions. Furthermore, the flow field and cell growth in this bioreactor were theoretically simulated. The results demonstrated that oscillatory shear stress significantly increased the viability and distribution uniformity of MSCs throughout the scaffold after culture for 3 weeks. Compared to static culture, oscillatory shear stress could promote the collagen secretion, mineral deposits, and osteogenic differentiation of MSCs. The findings obtained from this work indicate that the oscillatory perfusion not only provides a higher survival rate and a more uniform distribution of cells but also facilitates osteogenic differentiation of MSCs. Oscillating perfusion bioreactor culture of MSCs in 3D scaffold with optimal matrix stiffness could offer an easy-to-use but efficient bioreactor for bone tissue engineering.

Abstract. Phospholipase C (PLC) is a pivotal enzyme in the phosphoinositide pathway that promotes the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), to participate in eukaryotic signal transduction. Several PLC isozymes are associated with cancer, such as PLC-β1, PLC-δ1, PLC-ε and PLC-γ1. However, the role of PLC-δ3 (PLCD3) in nasopharyngeal carcinoma (NPC) has not been investigated to date. In our previous study, we demonstrated that flotillin2 (Flot2) plays a pro-neoplastic role in NPC and is involved in tumour progression and metastasis. In the present study, we screened the interacting proteins of Flot2 using the yeast two-hybrid (Y2H) method and verified the interaction between PLCD3 and Flot2 by co-immunoprecipitation. We also investigated the biological functions of PLCD3 in NPC. Inhibition of PLCD3 expression impaired the malignant potential of 5-8F, a highly metastatic NPC cell line, by restraining its growth, proliferation, mobility and migration. The present study demonstrated that PLCD3 may be an oncogenic protein in NPC and that it plays an important role in the progression of NPC partially by interacting with Flot2.

Our data indicate that ATP-binding cassette subfamily C member 4 may be a useful molecular marker in predicting radiosensitivity, and a potential target in improving the response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients.

The aim of this study was to evaluate the effects of maternal and dietary vitamin A ( VA ) level on growth performance, meat quality, antioxidant status, and immune function of offspring broilers. Chinese yellow-feathered breeder hens were fed a basal diet supplemented with 0, 5,400, 10,800, and 21,600 IU/kg VA for 8 wk, with 6 replicates of 22 hens per replicate. Then the offspring hatched from each of the 4 maternal groups were fed a basal diet supplemented with 0 or 5,000 IU/kg VA for 63 D. Overall, there were 8 treatment combinations, each with 6 replicate pens of 20 birds. Results showed that (1) providing VA in offspring diets increased final body weight ( FW ), average daily gain, and average daily feed intake but reduced feed-to-gain ratio and mortality of offspring broilers ( P < 0.05), whereas maternal provision of VA did not significantly affect the growth performance and mortality of offspring broilers. Maternal or offspring VA did not affect proportion of breast or thigh muscle ( P > 0.05). (2) Maternal feeding with 21,600 IU/kg VA increased ( P < 0.05) pH 24 h postmortem of breast muscle, compared with those without maternal supplication of VA. Dietary provision of 5,000 IU/kg VA in the posthatching diet decreased ( P < 0.05) drip loss, yellowness (b∗) value and lightness (L∗) value, and increased shear force and pH of breast muscle compared with those without dietary VA supplication. (3) Maternal or offspring VA did not affect the activities of total superoxide dismutase and glutathione peroxidase ( GSH-Px ) or the content of malondialdehyde; however, there was a significant interaction ( P < 0.05) between maternal and offspring VA on the activity of GSH-Px in serum. (4) Dietary provision of 5,000 IU/kg VA increased ( P < 0.05) the weight proportion of liver and bursa of fabricius, whereas maternal feeding with 21,600 IU/kg VA increased the hatchling BW. Maternal feeding with 5,400 and 21,600 IU/kg VA decreased ( P < 0.05) splenic interferon-γ ( IFN - γ ) transcripts and increased ( P < 0.05) those of interleukin-2 ( IL - 2 ) in the progeny. There were interactions ( P < 0.05) between maternal and offspring VA on splenic IL-2 , IL-1β, and IFN-γ expression. In summary, maternal and offspring provision of VA both had influence on meat quality and immune function in progeny broilers. Dietary VA increased growth performance, whereas the maternal VA affected the initial body weight of progeny when hatched, but the difference in performance caused by maternal VA level was able to be eliminated by dietary VA supplementation. Therefore, offspring provision had greater imp...

Background Cancer-associated cachexia is a multifactorial syndrome defined by progressive weight loss with ongoing loss of adipose tissue and skeletal muscle. Adipose loss occurs in the early stage of cachexia and is associated with reduced quality of life and survival time. Although numerous lncRNAs are regarded as novel regulators in adipose metabolism, the role of lncRNAs that selectively modulate the development of adipose loss in cachexia remains limited. Methods In this study, we analyzed microarray data of lncRNAs in adipose loss and further explored the function and mechanism of MALAT1 in adipose loss. First, we explored the expression and function of MALAT1 in adipose cell by quantitative PCR and RNA knockdown. Subsequently, the mechanism of MALAT1 involvement in adipose loss was analyzed via RNA-seq, bioinformatics analysis and reporter gene assay. Finally, we explored the clinical significance of MALAT1 through correlation analysis. Results Cellular experiments revealed that knocking down MALAT1 significantly inhibited the process of adipogenesis. RNA-seq data showed that numerous adipogenic genes were downregulated upon MALAT1 knockdown. A protein–protein interaction network analysis identified PPAR-γ as the central node transcription factor, the inhibition of which explains the downregulation of numerous adipogenic genes. A reporter gene assay suggested that MALAT1 can regulate the gene expression of PPAR-γ at the transcriptional level. Moreover, MALAT1 was weakly expressed in the subcutaneous white adipose tissue of cancer-associated cachexia patients and was related to low fat mass index and poor prognosis in cancer patients. Conclusions This study indicated that MALAT1 is associated with adipose loss in cancer-associated cachexia by regulating adipogenesis through PPAR-γ, which may potentially be a novel target for the diagnosis and treatment of cancer-associated cachexia in the clinic.