Limits...
Measuring Food Intake and Nutrient Absorption in Caenorhabditis elegans.

Gomez-Amaro RL, Valentine ER, Carretero M, LeBoeuf SE, Rangaraju S, Broaddus CD, Solis GM, Williamson JR, Petrascheck M - Genetics (2015)

Bottom Line: Caenorhabditis elegans has emerged as a powerful model to study the genetics of feeding, food-related behaviors, and metabolism.We show that serotonin-increased feeding leads to increased protein synthesis in a SER-7-dependent manner, including proteins known to promote aging.Protein content in the food has recently emerged as critical factor in determining how food composition affects aging and health.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

No MeSH data available.


Measurement of nutrient absorption in C. elegans using metabolic labeling coupled with quantitative mass spectrometry. To metabolically label worms, (A) we first grew three different OP50 “foods” by culturing OP50 bacteria in minimal media enriched with either 100% (14NH4)2SO4, 50% (14NH4)2SO4 + 50% (15NH4)2SO4, or 100% (15NH4)2SO4. These foods were termed “light,” “medium,” and “heavy,” respectively. Second, we generated a population of heavy worms. These worms were fed heavy OP50 for three generations to ensure fully enriched 100% (15NH4)2SO4 worm proteins. (B) Third, light and heavy worms were synchronized and cultured in light and heavy food, respectively. The heavy worms were used as an external standard to facilitate the comparison of different experimental samples. 14N worms were given either the drug of choice or water at day 1. At day 5, or the start day for the pulse labeling, worms were washed and the food was switched from light to medium for drug- or water-treated worms, and from heavy to heavy for the mass-spec-standard worms. The worms were harvested on day 7 after pulse labeling and prepared for mass spectrometry analysis. (C) Preparation of samples for mass spectrometry analysis. For each experiment an external standard was generated, derived from heavy worms cultured in parallel. Each sample lysate was spiked with the external standard. Proteins were extracted by TCA precipitation, digested with trypsin, and analyzed by LC-MS/MS.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4492371&req=5

fig4: Measurement of nutrient absorption in C. elegans using metabolic labeling coupled with quantitative mass spectrometry. To metabolically label worms, (A) we first grew three different OP50 “foods” by culturing OP50 bacteria in minimal media enriched with either 100% (14NH4)2SO4, 50% (14NH4)2SO4 + 50% (15NH4)2SO4, or 100% (15NH4)2SO4. These foods were termed “light,” “medium,” and “heavy,” respectively. Second, we generated a population of heavy worms. These worms were fed heavy OP50 for three generations to ensure fully enriched 100% (15NH4)2SO4 worm proteins. (B) Third, light and heavy worms were synchronized and cultured in light and heavy food, respectively. The heavy worms were used as an external standard to facilitate the comparison of different experimental samples. 14N worms were given either the drug of choice or water at day 1. At day 5, or the start day for the pulse labeling, worms were washed and the food was switched from light to medium for drug- or water-treated worms, and from heavy to heavy for the mass-spec-standard worms. The worms were harvested on day 7 after pulse labeling and prepared for mass spectrometry analysis. (C) Preparation of samples for mass spectrometry analysis. For each experiment an external standard was generated, derived from heavy worms cultured in parallel. Each sample lysate was spiked with the external standard. Proteins were extracted by TCA precipitation, digested with trypsin, and analyzed by LC-MS/MS.

Mentions: A complete understanding of feeding not only requires us to measure the amount of food eaten, but also the amount incorporated into the animal. To measure food absorption, we developed the pulse-feeding assay. In the pulse-feeding assay, a 15N pulse is delivered to C. elegans by feeding nitrogen-isotope-labeled (14N, 15N) bacteria (OP50). Nutrient (i.e., food) absorption is subsequently determined by measuring isotope incorporation into the C. elegans proteome, which is proportional to the bacteria ingested (see Supporting InformationDiscussion). To conduct such an experiment, C. elegans are raised on 14N-labeled bacteria. Then, 14N-labeled bacteria are exchanged with 50% 14N/50% 15N-labeled OP50 and fed to the worm throughout a period of interest (pulse interval). At the end of the pulse interval, proteins are extracted and the amount of incorporated 15N in the C. elegans proteome is determined by mass spectrometry (Figure 4, A and B).


Measuring Food Intake and Nutrient Absorption in Caenorhabditis elegans.

Gomez-Amaro RL, Valentine ER, Carretero M, LeBoeuf SE, Rangaraju S, Broaddus CD, Solis GM, Williamson JR, Petrascheck M - Genetics (2015)

Measurement of nutrient absorption in C. elegans using metabolic labeling coupled with quantitative mass spectrometry. To metabolically label worms, (A) we first grew three different OP50 “foods” by culturing OP50 bacteria in minimal media enriched with either 100% (14NH4)2SO4, 50% (14NH4)2SO4 + 50% (15NH4)2SO4, or 100% (15NH4)2SO4. These foods were termed “light,” “medium,” and “heavy,” respectively. Second, we generated a population of heavy worms. These worms were fed heavy OP50 for three generations to ensure fully enriched 100% (15NH4)2SO4 worm proteins. (B) Third, light and heavy worms were synchronized and cultured in light and heavy food, respectively. The heavy worms were used as an external standard to facilitate the comparison of different experimental samples. 14N worms were given either the drug of choice or water at day 1. At day 5, or the start day for the pulse labeling, worms were washed and the food was switched from light to medium for drug- or water-treated worms, and from heavy to heavy for the mass-spec-standard worms. The worms were harvested on day 7 after pulse labeling and prepared for mass spectrometry analysis. (C) Preparation of samples for mass spectrometry analysis. For each experiment an external standard was generated, derived from heavy worms cultured in parallel. Each sample lysate was spiked with the external standard. Proteins were extracted by TCA precipitation, digested with trypsin, and analyzed by LC-MS/MS.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492371&req=5

fig4: Measurement of nutrient absorption in C. elegans using metabolic labeling coupled with quantitative mass spectrometry. To metabolically label worms, (A) we first grew three different OP50 “foods” by culturing OP50 bacteria in minimal media enriched with either 100% (14NH4)2SO4, 50% (14NH4)2SO4 + 50% (15NH4)2SO4, or 100% (15NH4)2SO4. These foods were termed “light,” “medium,” and “heavy,” respectively. Second, we generated a population of heavy worms. These worms were fed heavy OP50 for three generations to ensure fully enriched 100% (15NH4)2SO4 worm proteins. (B) Third, light and heavy worms were synchronized and cultured in light and heavy food, respectively. The heavy worms were used as an external standard to facilitate the comparison of different experimental samples. 14N worms were given either the drug of choice or water at day 1. At day 5, or the start day for the pulse labeling, worms were washed and the food was switched from light to medium for drug- or water-treated worms, and from heavy to heavy for the mass-spec-standard worms. The worms were harvested on day 7 after pulse labeling and prepared for mass spectrometry analysis. (C) Preparation of samples for mass spectrometry analysis. For each experiment an external standard was generated, derived from heavy worms cultured in parallel. Each sample lysate was spiked with the external standard. Proteins were extracted by TCA precipitation, digested with trypsin, and analyzed by LC-MS/MS.
Mentions: A complete understanding of feeding not only requires us to measure the amount of food eaten, but also the amount incorporated into the animal. To measure food absorption, we developed the pulse-feeding assay. In the pulse-feeding assay, a 15N pulse is delivered to C. elegans by feeding nitrogen-isotope-labeled (14N, 15N) bacteria (OP50). Nutrient (i.e., food) absorption is subsequently determined by measuring isotope incorporation into the C. elegans proteome, which is proportional to the bacteria ingested (see Supporting InformationDiscussion). To conduct such an experiment, C. elegans are raised on 14N-labeled bacteria. Then, 14N-labeled bacteria are exchanged with 50% 14N/50% 15N-labeled OP50 and fed to the worm throughout a period of interest (pulse interval). At the end of the pulse interval, proteins are extracted and the amount of incorporated 15N in the C. elegans proteome is determined by mass spectrometry (Figure 4, A and B).

Bottom Line: Caenorhabditis elegans has emerged as a powerful model to study the genetics of feeding, food-related behaviors, and metabolism.We show that serotonin-increased feeding leads to increased protein synthesis in a SER-7-dependent manner, including proteins known to promote aging.Protein content in the food has recently emerged as critical factor in determining how food composition affects aging and health.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California 92037.

No MeSH data available.