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Browsing by Subject "Adenine nucleotides"
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Item Contribution of rankl regulation to bone resorption induced by PTH receptor activation in osteocytes(2012-10-19) Ben-awadh, Abdullah Nasser; Bellido, Teresita M.; Plotkin, Lilian I.; Allen, Matthew R.PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton.Item Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle(Elsevier, 2020-07) Davis, Patrick R.; Miller, Spencer G.; Verhoeven, Nicolas A.; Morgan, Joshua S.; Tulis, David A.; Witczak, Carol A.; Brault, Jeffrey J.; Anatomy and Cell Biology, School of MedicineBackground: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. Methods: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. Results: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. Conclusions: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.Item Liquid Chromatography Method for Simultaneous Quantification of ATP and its Degradation Products Compatible with both UV-Vis and Mass Spectrometry(Elsevier, 2022) Law, Andrew S.; Hafen, Paul S.; Brault, Jeffrey J.; Anatomy, Cell Biology and Physiology, School of MedicineATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV-Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV-Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV-Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV-Vis and their confirmation and/or identification of unknown peaks via MS.