Dataset Information

Amino acid and heavy water labelling to obtain protein turnover rates in intact adult animals

ABSTRACT: Both labelling studies were conducted with C57/BL6J adult male mice, derived from the same supplier and maintained under identical conditions. Eleven adult male C57BL/6JOlaHsd mice (Harlan UK Ltd., Shardlow, UK) of over 12 months of age at the start of the experiment were housed individually in 48 x 15 x 15 cm polypropylene cages (NKP Cages Ltd., Coalville, UK). Each cage contained substrate (Corn Cob Absorb 10-14 substrate), paper wool nest material and environmental enrichment. Food (LabDiet 5002 Certified Rodent Diet, Purina Mills, St. Louis, USA) and water were provided ad libitum. The mice were maintained on a reversed photo-period (light, 12 h; dark, 12 h; lights on at 2000 hrs) and at 19-21 oC ± 2 oC. Standard laboratory diet was replaced with a semisynthetic diet with the inclusion of [13C6]lysine at a relative isotope abundance (RIA) of 0.5. The dietary pellets were dissociated with water containing the dissolved [13C6]lysine to form a thick paste and mixed extensively. Once homogeneous, the paste was then extruded into strips 1 cm across and dried in a commercial foodstuff drying oven at 40 oC. The mice had access to the labelled diet for varying amounts of time: 0, 1, 2, 3, 4, 6, 9, 12, 17, 22 or 30 days. The day that the animals were introduced to the labelled diet was staggered for all culls and dissections to take place on the same day. All mice were humanely killed on day 30 and dissected to recover liver, kidney, heart and pooled hindlimb skeletal muscle from each animal. All resected tissue was frozen at -80 oC prior to analysis. For the heavy water labelling protocol, all animals were provided free access to LabDiet 5002. At the start of the experiment, mice were injected with two successive 0.5 mL injections, 4 hours apart, of 0.15 M sodium chloride dissolved in deuterated water. Thereafter, mice were given free access to 8% (v/v) [2H2]O for the duration of the experiment. After 0, 1, 2, 3, 6, 7, 9, 13, 16, 21, 24 and 30 days, mice were killed and dissected exactly as described for the amino acid labelling experiment and tissues were stored at -80 oC prior to analysis.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Robert J Beynon

PROVIDER: PXD029639 | JPOST Repository | Mon Dec 13 00:00:00 GMT 2021

REPOSITORIES: jPOST

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Publications

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Hammond Dean E DE Simpson Deborah M DM Franco Catarina C Wright Muelas Marina M Waters John J Ludwig R W RW Prescott Mark C MC Hurst Jane L JL Beynon Robert J RJ Lau Edward E

Molecular & cellular proteomics : MCP 20220528 7

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in po ...[more]

PMID: 35636728

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Project description:To study the molecular mediators of naturally rewarding effects of palatable food we used a model of palatable âsnackingâ (Ulrich-Lai et al., 2007) in which rats are given chronic, brief access to a limited amount of sucrose solution (30%). Single housed, male Long-Evans rats (250g) (n=12 per group) from Harlan Labs (Indianapolis, IN) received normal rat chow (Harlan Teklad) and water ad libitum for the duration of the experiment. After a one-week period of acclimation, rats were randomly assigned to drink treatment groups of either 30% sucrose solution or water. Rats received a 14-day regimen of twice daily (9:30 and 15:30) brief (maximum of 30 minutes) limited (up to 4 mL) access of their assigned drink solution. Drink solutions were delivered via a graduated sipper placed onto the cage top in addition to the existing water bottle and sippers were immediately removed when the animal had consumed 4mL or after the 30-minute access period, whichever occurred first. Drink intake, food intake, and body weight were monitored throughout the experiment to verify that the rats learned to drink sucrose, that they adjusted chow intake for calories consumed from sucrose (~10%), and that there was no effect on body weight gain as is normally seen with this model (Ulrich-Lai et al., 2007). Drink treatment terminated on day 14 and at 8:00 on the morning of day 15, the rats were sacrificed by rapid decapitation. BLA tissue was dissected, RNA extracted, and gene expression changes between water and sucrose groups were accessed by microarray. We used microarray to assess the changes in gene expression in the BLA of rats that have had a history of sucrose snacks (n=12) vs control (n=12). Rats received access (up to 4 mL) to extra bottles containing water (control) or 30% sucrose twice daily for 14 days. Rats had ab lib access to chow and water for the duration of the experiment. Rats were sacrificed the morning of day 15 and the BLA was removed for microarray analysis. Functional cluster analysis was performed on the list of 145 significantly upregulated genes using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA). See supplementary file at the foot of this record.

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