Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

BACKGROUND: The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis RESULTS: Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucine CONCLUSION: In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway

Synergism between cAMP and PPARγ signalling in the initiation of UCP1 gene expression in HIB1B brown adipocytes

Expression of the brown adipocyte-specific gene, uncoupling protein 1 (UCP1), is increased by both PPAR stimulation and cAMP activation through their ability to stimulate the expression of the PPAR coactivator PGC1. In HIB1B brown preadipocytes, combination of the PPAR agonist, rosiglitazone, and the cAMP stimulator forskolin synergistically increased UCP1 mRNA expression, but PGC1 expression was only increased additively by the two drugs. The PPAR antagonist, GW9662, and the PKA inhibitor, H89, both inhibited UCP1 expression stimulated by rosiglitazone and forskolin but PGC1 expression was not altered to the same extent. Reporter studies demonstrated that combined rosiglitazone and forskolin synergistically activated transcription from a full length 3.1 kbp UCP1 luciferase promoter construct, but the response was only additive and much reduced when a minimal 260 bp proximal UCP1 promoter was examined. Rosiglitazone and forskolin in combination were able to synergistically stimulate promoters comprising of tandem repeats of either PPREs or CREs. We conclude that rosiglitazone and forskolin act together to synergistically activate the UCP1 promoter directly rather than by increasing PGC1 expression and by a mechanism involving cross-talk between the signalling systems regulating the CRE and PPRE on the promoters

Cross-Product Extensions of the Gene Ontology

The Gene Ontology is being normalized and extended to include computable logical definitions. These definitions are partitioned into mutually exclusive cross-product sets, many of which reference other OBO Foundry ontologies. The results can be used to reason over the ontology, and to make cross-ontology queries

Gene expression of inflammatory markers in adipose tissue between obese women with polycystic ovary and normal obese women

OBJECTIVE: The pathogenesis of polycystic ovary syndrome (PCOS), a common endocrine disease and metabolic disturbance, is still unknown. The aim of the study was to investigate whether patients with PCOS display increased expression of inflammatory markers in adipose tissue. PATIENTS AND METHODS: Two groups of women were investigated, those diagnosed with PCOS (n = 8) and age and BMI-matched normal women (n = 12). Their age was between 20-45 years and all subjects were apparently healthy and did not take any medications. Adipose tissue levels of mRNA of inflammatory markers were determined by use of real-time PCR. RESULTS: There were no differences between obese patients and obese PCOS in levels of adipocytokines. CONCLUSIONS: There were no effects of PCOS on the expression of any of the adipocytokines genes measured in subcutaneous adipose tissue

A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue

Peer reviewedPublisher PD

C/EBP␤ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression *

cAMP-dependent protein kinase induction of PPAR␥ coactivator-1␣ (PGC-1␣) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1␣ in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1␣ and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1␣ reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBP␤, but not C/EBP␣ and C/EBP␦, bound to the CRE on the PGC-1␣ promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBP␤ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBP␤ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBP␤ rescued cAMP-inducible PGC-1␣ and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBP␤ on the CRE in PGC-1␣ proximal promoter

Py-GC/MS applied to the analysis of synthetic organic pigments: characterization and identification in paint samples

A collection of 76 synthetic organic pigments was analysed using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS). The purpose of this work was to expand the knowledge on synthetic pigments and to assess characteristic pyrolysis products that could help in the identification of these pigments in paint samples. We analysed several classes of synthetic pigments not previously reported as being analysed by this technique: some metal complexes, β-naphthol pigment lakes, BONA pigment lakes, disazopyrazolone, triarylcarbonium, dioxazine, anthraquinone, indanthrone, isoindoline and thioindigo classes. We also report for the first time the Py-GC/MS analysis of a number of naphthol AS, benzimidazolone, phthalocyanine and perylene pigments and other miscellaneous pigments including pigments with unpublished chemical structure. We successfully used the Py-GC/MS technique for the analysis of paints by artists Clyfford Still and Jackson Pollock to identify the synthetic organic pigments and the binding media

Comparing radiolytic production of H2O2 and development of Zebrafish embryos after ultra high dose rate exposure with electron and transmission proton beams.

The physico-chemical and biological response to conventional and UHDR electron and proton beams was investigated, along with conventional photons. The temporal structure and nature of the beam affected both, with electron beam at ≥1400 Gy/s and proton beam at 0.1 and 1260 Gy/s found to be isoefficient at sparing zebrafish embryos

The State of the Region: Hampton Roads 2012

This is Old Dominion University\u27s 13th annual State of the Region Report. While it represents the work of many people connected in various ways to the university, the report does not constitute an official viewpoint of Old Dominion or it\u27s president, John R. Broderick. The report maintains the goal of stimulating thought and discussion that ultimately will make Hampton Roads an even better place to live. We are proud of our region\u27s many successes, but realize that it is possible to improve our performance. In order to do so, we must have accurate information about where we are and a sound understanding of the policy options open to us.https://digitalcommons.odu.edu/economics_books/1006/thumbnail.jp