Effects of leucine supplementation and resistance exercise on dexamethasone-induced muscle atrophy and insulin resistance in rats

Objective: We aimed to evaluate the effects of resistance exercise (RE) and leucine (LEU) supplementation on dexamethasone (DEXA)-induced muscle atrophy and insulin resistance. Methods: Male Wistar rats were randomly divided into DEXA(DEX), DEXA + RE (DEX-RE), DEXA + LEU (DEX-LEU), and DEXA + RE + LEU (DEX-RE-LEU) groups. Each group received DEXA 5 mg . kg(-1) . d(-1) for 7 d from drinking water and were pair-fed to the DEX group; LEU-supplemented groups received 0.135 g . kg(-1) . d(-1) through gavage for 7 d; the RE protocol was based on three sessions of squat-type exercise composed by three sets of 10 repetitions at 70% of maximal voluntary strength capacity. Results: The plantaris mass was significantly greater in both trained groups compared with the non-trained groups. Muscle cross-sectional area and fiber areas did not differ between groups. Both trained groups displayed significant increases in the number of intermediated fibers (IIa/IIx), a decreased number of fast-twitch fibers (IIb), an increased ratio of the proteins phospho(Ser2448)/ total mammalian target of rapamycin and phospho(Thr389)/total 70-kDa ribosomal protein S6 kinase. and a decreased ratio of phospho(Ser253)/total Forkhead box protein-3a. Plasma glucose was significantly increased in the DEX-LEU group compared with the DEX group and RE significantly decreased hyperglycemia. The DEX-LEU group displayed decreased glucose transporter-4 translocation compared with the DEX group and RE restored this response. LEU supplementation worsened insulin sensitivity and did not attenuate muscle wasting in rats treated with DEXA. Conversely, RE modulated glucose homeostasis and fiber type transition in the plantaris muscle. Conclusion: Resistance exercise but not LEU supplementation promoted fiber type transition and improved glucose homeostasis in DEXA-treated rats. (C) 2012 Elsevier Inc. All rights reserved.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.