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Protocol for the measurement of fatty acid and glycerol turnover in vivo in baboons.

ABSTRACT: Recognition of the strength of nonhuman primate models in investigating metabolic disorders has resulted in an expanded need for in vivo research techniques. We studied adipose metabolism in 10 baboons (13.0 ± 4.2 years old, 29.5 ± 5.5 kg). Part 1 evaluated the effect of different sedatives on the rate of appearance of plasma free fatty acids (RaFFA), assessed using ¹³C?-labeled palmitate infusion (7 µmol/kg/min). Animals, were studied with no sedation, with complete isoflurane sedation, and with minimal midazolam infusion (0.04 mg/kg/h), with the last scheme allowing for the most consistent values and animals that were visually more calm. In Part 2, RaFFA and RaGlycerol (D?-glycerol, 5 mg/kg lean body mass/h) were measured. From midnight to 0300, flux fell and came to a steady state between 0500 and 0700 h (RaFFA, 39.4 ± 29.8 ?mol/kg fat mass/min; and RaGlycerol, 26.9 ± 7.3 ?mol/kg/min). The RaFFA-to-RaGlycerol ratio was 1.5 ± 0.8 (49% reesterification). The decline in turnover throughout the night reflects natural circadian processes and was mirrored by reductions in FFA and glycerol to 0.62 and ± 0.14 and 0.16 and ± 0.03 mmol/l, respectively. The concurrent changes in both FFA and glycerol kinetics indicate physiologic validity of the method. These techniques will support needed research to determine mechanisms by which treatments act upon the adipocyte in vivo.

SUBMITTER: Bastarrachea RA

PROVIDER: S-EPMC3090248 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Protocol for the measurement of fatty acid and glycerol turnover in vivo in baboons.

Bastarrachea Raul A RA Veron Sonya M SM Vaidyanathan Vidya V Garcia-Forey Maggie M Voruganti V Saroja VS Higgins Paul B PB Parks Elizabeth J EJ

Journal of lipid research 20110317 6

Recognition of the strength of nonhuman primate models in investigating metabolic disorders has resulted in an expanded need for in vivo research techniques. We studied adipose metabolism in 10 baboons (13.0 ± 4.2 years old, 29.5 ± 5.5 kg). Part 1 evaluated the effect of different sedatives on the rate of appearance of plasma free fatty acids (RaFFA), assessed using ¹³C₄-labeled palmitate infusion (7 µmol/kg/min). Animals, were studied with no sedation, with complete isoflurane sedation, and wit ...[more]

PMID: 21415122

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Project description:Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (?fadBA145?glpD, ?fadBA145?betAB, and ?fadBA145?betAB?glpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection.

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Protocol for the measurement of fatty acid and glycerol turnover in vivo in baboons.

Publications

Protocol for the measurement of fatty acid and glycerol turnover in vivo in baboons.

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