Whey protein intake after resistance exercise activates mTOR signaling in a dose-dependent manner in human skeletal muscle

Purpose

Protein ingestion after resistance exercise increases muscle protein synthesis (MPS) in a dose-dependent manner. However, the molecular mechanism(s) for the dose-dependency of MPS remains unclear. This study aimed to determine the dose response of mammalian target of rapamycin (mTOR) signaling in muscle with ingestion of protein after resistance exercise.

Methods

Fifteen male subjects performed four sets of six unilateral isokinetic concentric knee extensions. Immediately after exercise, eight subjects consumed water only. The other seven subjects, in a randomized-order crossover design, took either a 10 [3.6 g essential amino acids (EAA)] or 20 g (7.1 g EAA) solution of whey protein. Muscle biopsies from the vastus lateralis muscle were taken 30 min before and 1 h after resistance exercise. Phosphorylation of Akt (Ser473), mTOR (Ser2448), 4E-BP1 (Thr37/46), and S6K1 (Thr389) was measured by western blotting.

Results

Concentric knee extension exercise alone did not increase phosphorylation of Akt and mTOR 1 h after exercise, but ingesting protein after exercise significantly increased the phosphorylation of Akt and mTOR in a dose-dependent manner (P < 0.05). 4E-BP1 phosphorylation significantly decreased after resistance exercise (P < 0.05), but subjects who took 10 or 20 g of protein after exercise showed increased 4E-BP1 from post-exercise dephosphorylation (P < 0.05). S6K1 phosphorylation significantly increased after resistance exercise (P < 0.05), and 20 g of protein further increased S6K1 phosphorylation compared with ingestion of 10 g (P < 0.05).

Conclusions

These findings suggest that whey protein intake after resistance exercise activates mTOR signaling in a dose-dependent manner in untrained men.     

Continuous fever-range heat stress induces thermotolerance in odontoblast-leneage cells

Objective

Heat shock during restorative procedures can trigger damage to the pulpodentin complex. While severe heat shock has toxic effects, fever-range heat stress exerts beneficial effects on several cells and tissues. In this study, we examined whether continuous fever-range heat stress (CFHS) has beneficial effects on thermotolerance in the rat clonal dental pulp cell line with odontoblastic properties, KN-3.

Methods

KN-3 cells were cultured at 41 °C for various periods, and the expression level of several proteins was assessed by Western blot analysis. After pre-heat-treatment at 41 °C for various periods, KN-3 cells were exposed to lethal severe heat shock (LSHS) at 49 °C for 10 min, and cell viability was examined using the MTS assay. Additionally, the expression level of odontoblast differentiation makers in surviving cells was examined by Western blot analysis.

Results

CFHS increased the expression levels of several heat shock proteins (HSPs) in KN-3 cells, and induced transient cell cycle arrest. KN-3 cells, not pre-heated or exposed to CFHS for 1 or 3 h, died after exposure to LSHS. In contrast, KN-3 cells exposed to CFHS for 12 h were transiently lower on day 1, but increased on day 3 after LSHS. The surviving cells expressed odontoblast differentiation markers, dentine sialoprotein and dentine matrix protein-1. These results suggest that CFHS for 12 h improves tolerance to LSHS by inducing HSPs expression and cell cycle arrest in KN-3 cells.

Conclusions

The appropriate pretreatment with continuous fever-range heat stress can provide protection against lethal heat shock in KN-3 cells.     

Hyperthermia generated with ferucarbotran (Resovist®) in an alternating magnetic field enhances cisplatin-induced apoptosis of cultured human oral cancer cells

Hyperthermia is a promising anti-cancer treatment in which the tissue temperature is increased to 42–45 °C, and which is often used in combination with chemotherapy or radiation therapy. Our aim in the present work was to examine the feasibility of combination therapy for oral cancer with cisplatin and hyperthermia generated with ferucarbotran (Resovist^®; superparamagnetic iron oxide) in an alternating magnetic field (AMF). First, we established that administration of ferucarbotran at the approved dosage for magnetic resonance imaging provides an iron concentration sufficient to increase the temperature to 42.5 °C upon exposure to AMF. Then, we examined the effect of cisplatin combined with ferucarbotran/AMF-induced hyperthermia on cultured human oral cancer cells (HSC-3 and OSC-19). Cisplatin alone induced apoptosis of cancer cells in a dose-dependent manner, as is well known. However, the combination of cisplatin with ferucarbotran/AMF was significantly more effective than cisplatin alone. This result suggests that it might be possible to reduce the clinically effective dosage of cisplatin by administering it in combination with ferucarbotran/AMF-induced hyperthermia, thereby potentially reducing the incidence of serious cisplatin-related side effects. Further work seems justified to evaluate simultaneous thermo-chemotherapy as a new approach to anticancer therapy.     

Nematode ascarosides attenuate mammalian type 2 inflammatory responses

Mounting evidence suggests that nematode infection can protect against disorders of immune dysregulation. Administration of live parasites or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for immune disorders, including asthma. Human clinical trials of live parasite ingestion for the treatment of immune disorders have produced promising results, yet concerns persist regarding the ingestion of pathogenic organisms and the immunogenicity of protein components. Despite extensive efforts to define the active components of ES products, no small molecules with immune regulatory activity have been identified from nematodes. Here we show that an evolutionarily conserved family of nematode pheromones called ascarosides strongly modulates the pulmonary immune response and reduces asthma severity in mice. Screening the inhibitory effects of ascarosides produced by animal-parasitic nematodes on the development of asthma in an ovalbumin (OVA) murine model, we found that administration of nanogram quantities of ascr#7 prevented the development of lung eosinophilia, goblet cell metaplasia, and airway hyperreactivity. Ascr#7 suppressed the production of IL-33 from lung epithelial cells and reduced the number of memory-type pathogenic Th2 cells and ILC2s in the lung, both key drivers of the pathology of asthma. Our findings suggest that the mammalian immune system recognizes ascarosides as an evolutionarily conserved molecular signature of parasitic nematodes. The identification of a nematode-produced small molecule underlying the well-documented immunomodulatory effects of ES products may enable the development of treatment strategies for allergic diseases.

Parasitic nematodes are associated with almost all groups of vertebrates, and nearly one-third of the human population is infected with these helminths (1). Their omnipresence is in part due to their ability to modulate host immune responses to prevent immune attack and expulsion (2). The elimination of nematode infections has been proposed as a possible cause of the increased incidence of autoimmune disorders and allergic diseases in developed countries (3), based on epidemiological data showing a correlation between the decline in helminth infection and the rise in allergic and autoimmune diseases, including asthma, multiple sclerosis (MS), type 1 diabetes, and inflammatory bowel diseases (IBDs) (4).The administration of live nematodes or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for these immune disorders (5–8). The US Food and Drug Administration recently approved live helminth administration as an investigational drug for the treatment of immune disorders, and relevant human clinical trials are ongoing (9). Despite mounting evidence that helminths have significant therapeutic potential, we do not yet have a comprehensive understanding of the molecules that underlie their immunomodulatory effects; and, in particular, the possible relevance of low-molecular-weight components of ES products has remained largely unexplored.A wide range of nematodes, including many parasitic species, produce ascarosides, a family of small-molecule signals based on glycosides of the dideoxysugar ascarylose (10). Ascarosides have not yet been identified in any other animal phylum, suggesting that they may be a nematode-specific class of small molecules (SI Appendix, Fig. S1A). The first ascaroside-based signaling molecules were identified in the free-living model nematode Caenorhabditis elegans (11, 12). Ascarosides regulate almost every aspect of C. elegans life history, including diapause (dauer) induction, aging, mate finding, and aggregation (11, 12). Subsequently, ascarosides have been shown to be detected by organisms other than nematodes, such as nematophagous fungi that set traps to capture and digest nematodes (13). The perception of ascarosides is sufficient to trigger trap formation in these fungi, demonstrating their longstanding evolutionary association with nematodes. Furthermore, ascarosides produced by plant-pathogenic nematodes have been shown to trigger innate immune responses in monocot and dicot plants (14). Cumulatively, these findings suggest that ascarosides represent a nematode-specific molecular signature that is recognized and interpreted by nematode predators and hosts across multiple kingdoms.In this study, we collected ES products from the gut-resident, rodent-parasitic nematode Nippostrongylus brasiliensis. Previous studies showed that the administration of N. brasiliensis ES (NES) products fully inhibits the development of airway hyperresponsiveness (AHR) in the ovalbumin (OVA) murine model of asthma (15). Specifically, NES products substantially prevented lung eosinophilia, mucus production, and resistance to airflow. Notably, it was found that heat-treated or proteinase K–treated NES mimicked the full effect of untreated NES products in reducing lung eosinophilia and OVA-specific IgG in serum. Therefore, we hypothesized that the therapeutic effect of NES products may be due to the presence of specific small molecules that may in part be bound to secreted proteins, explaining the activity of heat- or proteinase K–treated NES. To test this hypothesis, we isolated the small molecule fraction of heat-treated NES (small molecule ES [smES]) products via filtration through a 3-kDa filter and found that smES products strongly suppresses OVA-induced allergic immune responses. Parallel chemical analyses of several other mammalian parasitic nematodes confirmed the presence of specific ascarosides in smES products of all tested species. Next, we tested synthetic samples of ascarosides and found that ascr#7, a compound produced by N. brasiliensis and other parasitic species, markedly inhibited the development of allergic airway inflammation, comparable to the full effect of smES products. Mechanistically, we found that ascr#7 administration attenuated IL-33 production from lung epithelial cells and suppressed the proliferation of memory-type IL-5–producing pathogenic T helper 2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) in the lung, both key drivers for the pathology of asthma. We thus demonstrate that ascarosides have an immunomodulatory role that attenuates OVA-induced allergic inflammation in a murine model.     

Adverse Tissue Reactions and Metal Ion Behavior After Small‐Head Metasul Hip Arthroplasty: A Long‐Term Follow‐Up Study

ObjectiveTo investigate the long‐term survivorship, incidence of adverse reactions to metal debris (ARMD), and metal ion behavior in patients who underwent small‐head Metasul metal‐on‐metal (MoM) total hip arthroplasty (THA).MethodsBetween February 1998 and September 2003, a retrospective study was performed on 43 consecutive patients (43 hips) who underwent unilateral cementless Metasul MoM THAs at our institution. Of them, 35 patients (nine males and 26 females) who were available for follow‐up more than 15 years after THA were enrolled in this study and underwent metal artifact reduction sequence magnetic resonance imaging (MARS‐MRI) to identify ARMD. The mean age at surgery of the patients was 59.7 years old (range, 31–83). Clinical and radiographic outcomes were evaluated retrospectively. Clinical examinations were conducted using the Harris Hip Score (HHS). Serum cobalt (Co) and chromium (Cr) ion levels and Co/Cr ratio were assessed at different postoperative periods of <5, 5–10, 11–14, and ≥15 years.ResultsThe mean follow‐up period for the 35 patients included was 18.1 years (range, 15–22). The mean HHS significantly improved from 44.6 ± 11.3 points preoperatively to 89.4 ± 7.9 points at the final follow‐up (P < 0.0001). ARMD was found in 20% of the patients using MARS‐MRI. No signs of stem loosening were found clinically or radiographically, whereas cup loosening and ARMD were observed in three patients (9%), for whom revision THAs were performed. The Kaplan–Meier survival rates with revision for any reason as the endpoint were 90.9% at 5 years, 84.8% at 10 years, 84.8% at 15 years (95% CI, 67.1–93.6), and 70.3% at 20 years (95% CI, 43.6–87.0). The survival rates with revision for ARMD as the endpoint were 100% at 5 years, 96.6% at 10 years, 96.6% at 15 years (95% CI, 77.2–99.7), and 80.1% at 20 years (95% CI, 45.3–95.2). Serum Co ion level peaked at 5–10 years after THA, which was significantly higher than that <5 years; however, it decreased to the initial level after 15 years. In contrast, serum Cr ion level significantly increased at 5–10 years and then remained almost constant. Significant differences in Cr ion levels (1.0 vs 2.0 μg/L, P = 0.024) and Co/Cr ratio (1.3 vs 0.9, P = 0.037) were found between non‐ARMD and ARMD patients at >11 years postoperatively.ConclusionOur results suggest that increased Cr ion levels and decreased Co/Cr ratio may be signs of ARMD in patients who underwent small‐head Metasul MoM THA.     

Hyperhomocyst(e)inemia impairs angiogenesis in a murine model of limb ischemia

Hyperhomocyst(e)inemia (HH) is an established independent risk factor for coronary, cerebral and peripheral vascular diseases. Recent studies have indicated that certain cardiovascular risk factors, including diabetes and hypercholesterolemia, impair expression of vascular endothelial growth factor (VEGF) and endogenous angiogenesis. In this study, we investigate the impact of moderate HH on angiogenesis and VEGF pathway in a mouse model of hindlimb ischemia. Upon induction of unilateral hindlimb ischemia, endogenous angiogenesis, expression of VEGF, and phosphorylation of the VEGF receptor Flk-1 were evaluated in mice heterozygous for a deletion of the cystathionine beta-synthase gene (CBS) and compared with those observed in CBS+/+ mice. CBS+/- mice exhibit moderate HH, as demonstrated by measuring plasma total homocyst(e)ine (tHcy) levels, which were significantly higher in these animals compared with CBS+/+ mice (4.77 +/- 0.82 vs 2.10 +/- 0.28, p < 0.01). Twenty-eight days after induction of ischemia, hindlimb blood flow was significantly reduced in CBS+/- mice compared with CBS+/+ animals (0.49 +/- 0.03, n = 12 vs 0.71 +/- 0.09, n = 10; p < 0.05). In addition, there was a significant negative correlation between plasma homocyst(e)ine levels and the laser Doppler perfusion ratio in CBS+/- mice (p = 0.0087, r = -0.7171). While VEGF expression and Flk-1 phosphorylation were not impaired in the ischemic muscles of CBS+/- mice, phosphorylation of the endothelial cell survival factor Akt was significantly inhibited by homocyst(e)ine in a dose-dependent manner in human umbilical vein endothelial cell (HUVECs) in vitro. In conclusion, our findings demonstrate that endogenous angiogenesis is inversely related to plasma levels of homocyst(e)ine in genetically engineered, heterozygous mice with moderate HH. This impairment, however, is not dependent on reduced expression of VEGF or impaired phosphorylation of its receptor Flk-1. In contrast, our data suggest that impaired Akt phosphorylation mediates the impairment of angiogenesis associated with HH.     

Mutation analysis of a Japanese patient with fucosidosis

Fucosidosis is a rare autosomal recessive disorder resulting from a deficiency of α-L-fucosidase. Recently, various mutations have been reported in this disease, but it is difficult to elucidate the phenotype from the genetic mutations. We report a patient with chronic infantile type fucosidosis, with a compound heterozygote of a nonsense mutation (W148X, Trp at codon 148 to stop codon) and a large deletion, including all exons. This is the first report of a large deletion demonstrated in fucosidosis. It is interesting that this patient has a relatively mild clinical course despite the absence of the mRNA. This case also indicates the difficulty in determining the phenotype from the genotype in fucosidosis. Received: February 19, 1999 / Accepted: April 16, 1999     

Inhibitor of apoptosis protein family as diagnostic markers and therapeutic targets of colorectal cancer

The apoptosis and antiapoptotic signaling pathways are important for regulating carcinogenesis and cancer progression, and for determining prognosis. Molecules involved in apoptosis represent potential cancer diagnostic markers and therapeutic targets. The inhibitor of apoptosis protein (IAP) family includes several important molecules involved in apoptosis that might represent such targets. Increasing evidence has demonstrated that the IAP family of proteins is integral for antiapoptotic and nuclear factor-κB signal transduction, and enhanced expression of IAPs contributes to colon carcinogenesis and its poor prognosis, as well as to drug resistance of tumors. X-linked IAP, cIAP1, cIAP2, and survivin are prognostic markers of colorectal cancer, and survivin and cIAP2 are also utilized to predict the effect of anticancer treatment in colorectal cancer patients. Novel therapies such as YM155 and LY2181308 targeting survivin, AEG35156 and phenoxodiol targeting X-linked IAP, AT-406 as a Smac mimetic, and survivin peptides are currently being evaluated in clinical trials. This report reviews the involvement of the IAP family in colorectal adenocarcinoma in order to summarize the role of the IAP family members as diagnostic and therapeutic targets, and to provide an overview of the future course of research in this area.