Preclinical Evaluation of a Food-Derived Functional Ingredient to Address Skeletal Muscle Atrophy.

Skeletal muscle is the metabolic powerhouse of the body, however, dysregulation of the mechanisms involved in skeletal muscle mass maintenance can have devastating effects leading to many metabolic and physiological diseases. The lack of effective solutions makes finding a validated nutritional intervention an urgent unmet medical need. In vitro testing in murine skeletal muscle cells and human macrophages was carried out to determine the effect of a hydrolysate derived from vicia faba (PeptiStrong: NPN_1) against phosphorylated S6, atrophy gene expression, and tumour necrosis factor alpha (TNF-α) secretion, respectively. Finally, the efficacy of NPN_1 on attenuating muscle waste in vivo was assessed in an atrophy murine model. Treatment of NPN_1 significantly increased the phosphorylation of S6, downregulated muscle atrophy related genes, and reduced lipopolysaccharide-induced TNF-α release in vitro. In a disuse atrophy murine model, following 18 days of NPN_1 treatment, mice exhibited a significant attenuation of muscle loss in the soleus muscle and increased the integrated expression of Type I and Type IIa fibres. At the RNA level, a significant upregulation of protein synthesis-related genes was observed in the soleus muscle following NPN_1 treatment. In vitro and preclinical results suggest that NPN_1 is an effective bioactive ingredient with great potential to prolong muscle health.

Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in the TSC1 or TSC2 genes and is characterized by widespread tumour growth, intractable epilepsy, cognitive deficits and autistic behaviour. CBD has been reported to decrease seizures and inhibit tumour cell progression, therefore we sought to determine the influence of CBD on TSC pathology in zebrafish carrying a nonsense mutation in the tsc2 gene. CBD treatment from 6 to 7 days post-fertilization (dpf) induced significant anxiolytic actions without causing sedation. Furthermore, CBD treatment from 3 dpf had no impact on tsc2-/- larvae motility nor their survival. CBD treatment did, however, reduce the number of phosphorylated rpS6 positive cells, and their cross-sectional cell size. This suggests a CBD mediated suppression of mechanistic target of rapamycin (mTOR) activity in the tsc2-/- larval brain. Taken together, these data suggest that CBD selectively modulates levels of phosphorylated rpS6 in the brain and additionally provides an anxiolytic effect. This is pertinent given the alterations in mTOR signalling in experimental models of TSC. Additional work is necessary to identify upstream signal modulation and to further justify the use of CBD as a possible therapeutic strategy to manage TSC.

Glucosamine activates autophagy in vitro and in vivo.

OBJECTIVE: Aging-associated changes in articular cartilage represent a main risk factor for osteoarthritis (OA). Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimentally induced defects in autophagy contribute to organismal- and tissue-specific aging, while enhancement of autophagy may protect against certain aging-related pathologies such as OA. The objective of this study was to determine whether glucosamine can activate autophagy. METHODS: Chondrocytes from normal human articular cartilage were treated with glucosamine (0.1- 10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3, and ribosomal protein S6 were determined by Western blotting. Autophagosome formation was analyzed by confocal microscopy. Reporter mice systemically expressing green fluorescent protein (GFP) fused to light chain 3 (LC3) (GFP-LC3-transgenic mice) were used to assess changes in autophagy in response to starvation and glucosamine treatment. RESULTS: Glucosamine treatment of chondrocytes activated autophagy, as indicated by increased LC3-II levels, formation of LC3 puncta, and increased LC3 turnover. This was associated with glucosamine-mediated inhibition of the Akt/FoxO3/mammalian target of rapamycin pathway. Administration of glucosamine to GFP-LC3-transgenic mice markedly activated autophagy in articular cartilage. CONCLUSION: Glucosamine modulates molecular targets of the autophagy pathway in vitro and in vivo, and the enhancement of autophagy is mainly dependent on the Akt/FoxO/mTOR pathway. These findings suggest that glucosamine is an effective autophagy activator and should motivate future studies on the efficacy of glucosamine in modifying aging-related cellular changes and supporting joint health.

Analysis of the pharmacodynamic activity of the mTOR inhibitor ridaforolimus (AP23573, MK-8669) in a phase 1 clinical trial.

PURPOSE: As part of a phase 1 dose-escalation trial, the pharmacodynamic activity of the mammalian target of rapamycin (mTOR) inhibitor ridaforolimus was assessed in multiple tissues by measuring levels of phosphorylated 4E binding protein-1 (p-4E-BP1) or S6, two downstream markers of mTOR activity. METHODS: 32 patients (pts) were dosed intravenously with ridaforolimus once daily for 5 consecutive days (QD × 5) every 2 weeks. The pharmacodynamic activity of ridaforolimus was assessed in peripheral blood mononuclear cells (PBMCs; 32 pts), skin (28 pts), and tumor specimens (3 pts) collected before and after dosing by measuring levels of p-4E-BP1 by immunoblot analysis or pS6 by immunohistochemistry. Levels of these markers were assessed in up to 19, 5, and 2 pre- and post-dose time points in PBMC, skin, and tumor specimens, respectively. RESULTS: In preclinical models, ridaforolimus induced a dose-dependent inhibition of p-4E-BP1 in PBMCs that was associated with antitumor activity. Rapid and potent inhibition of mTOR was observed in PBMCs from all 32 pts dosed, with a median level of inhibition of 96% observed within 1 h after the first dose. Inhibition of mTOR (>90%) was sustained during the entire QD × 5 dosing period, and substantial inhibition was still observed after the 9-day holiday between dosing courses. Evidence of mTOR inhibition was also obtained in skin in pts from all dose cohorts, although it did not persist through the break between courses. After two to three doses of ridaforolimus, inhibition of mTOR was detected in the tumor from one of three pts analyzed. CONCLUSIONS: Ridaforolimus was shown to inhibit its intended target, mTOR, in PBMCs, skin, and tumors. In PBMCs and skin, inhibition was observed at all dose levels tested, thus supporting but not driving the selection of a recommended phase 2 dose.

Polypyrimidine tract-binding protein is involved in regulation of albumin synthesis in response to food intake.

Our recent study demonstrates that polypyrimidine tract-binding protein (PTB), which is a sequence specific RNA-binding protein, attenuates albumin synthesis in a cell-free translation system. In this study, the effects of food intake on regulation of albumin synthesis through binding of PTB to albumin messenger RNA (mRNA) were investigated. Rats were divided into 1 of 3 groups: fed; fasted for 36 h; or fasted for 36 h and then refed for 24 h. No significant differences in albumin mRNA levels were found among fed, fasted and refed rats. However, a decrease in the proportion of albumin mRNA associated with polysomes was identified in fasted rats. Furthermore, UV-cross linking analysis demonstrated that levels of albumin mRNA-PTB complex were increased in liver extracts from fasted rats. No significant differences in PTB levels in liver homogenate were found among the experimental groups. However, PTB level in the cytoplasmic fraction was higher in fasted rats than in fed rats. In refed rats, PTB level in the cytoplasmic fraction returned to a level comparable to that in fed rats, but was inhibited by treatment with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. These results suggest that localization of PTB is regulated by food intake through mTOR signaling, and alterations in level of albumin mRNA-PTB complex play a role in mediating the effects of food intake on albumin synthesis in the rat liver.

Chemotherapy disrupts activity of translational regulatory proteins in bone marrow stromal cells.

OBJECTIVE: Bone marrow stromal cell function is a critical influence on hematopoietic reconstitution following progenitor or stem cell transplantation. Stromal cells support hematopoietic cell migration, survival, and proliferation. We have previously reported that stromal cell matrix metalloproteinase-2 (MMP-2) is necessary for optimal support of pro-B-cell chemotaxis through its regulation of stromal cell-derived factor-1 (CXCL12) release. Following exposure to the topoisomerase II inhibitor, etoposide (VP-16), stromal cell MMP-2 protein expression is reduced. The current study investigated the mechanism by which VP-16 may alter translation of MMP-2 in bone marrow stromal cells. MATERIALS AND METHODS: Bone marrow stromal cells were exposed to chemotherapeutic agents etoposide, melphalan, and 4-hydroperoxycyclophosphamide (4HC) and evaluated for MMP-2 expression by enzyme-linked immunosorbent assay and support of pro-B-cell chemotaxis by chemotaxis assay. Western blot analyses were completed to evaluate phosphorylation of stromal cell translational regulatory proteins 4E binding protein-1 (4EBP-1), P70(S6K), and S6 or MMP-2 in the presence of chemotherapy, or the chemical inhibitors rapamycin or LY294002. RESULTS: Rapid dephosphorylation of 4EBP-1, P70(S6K), and S6 following VP-16 exposure was observed, consistent with blunted translational efficiency. We also observed that inhibition of stromal cell mammalian target of rapamycin with rapamycin, or phosphatidylinositol 3 kinase with LY294002, resulted in inhibition of stromal cell MMP-2 protein. In addition we found that the chemotherapeutic agents melphalan and 4HC disrupt bone marrow stromal cell MMP-2 protein expression and support of chemotaxis. CONCLUSIONS: These data suggest that one mechanism by which chemotherapy may alter stromal cells of the bone marrow microenvironment is through disrupted translation of proteins.

Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents.

It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.