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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo lipogenesis in brown adipose tissue in vivo.

Abstract

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.

Introduction

Obesity and associated metabolic diseases are a pandemic that endanger present and future generations1,2. Commonly simplified as the consequence of the imbalance between energy intake and expenditure, the metabolic dysregulation associated with obesity affects a large number of metabolic pathways controlled by environmental and endogenous factors3. However, only a few pathways are routinely tested in animal models of metabolic dysregulation.

As an example, glucose disposal is routinely measured by glucose and insulin tolerance tests, probably due to the simpl....

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee at Cincinnati Children's Hospital Medical Center.

1. Preparation of D2O

NOTE: To avoid experimental variation, prepare sufficient solution/drinking water for all mice for the duration of the experiment.

  1. For intraperitoneal injection: generate 0.9% w/v saline D2O by dissolving 9 g of NaCl per liter of D2O. Filter through a.......

Representative Results

Based on the D2O dosing described in step 1, we typically find that body water is enriched in the range of 2.5% to 6%, and that a baseline level of deuterium enrichment in body water is rapidly achieved in 1 h and maintained for the duration of the study via 8% enriched drinking water (Figure 1). Continuous steady state body water enrichment is an assumption of the calculations used in step 6, and so we recommend experimental validation of the kinetics of body water enric.......

Discussion

Understanding the balance and interaction between complex metabolic pathways is an indispensable step toward understanding the biological basis of metabolic related diseases. Here, we show a noninvasive and inexpensive methodology to determine changes in de novo fatty acid synthesis. This method is adapted from previously published methods which developed calculations for estimating de novo synthesis flux from fatty acid deuterium enrichment31 and for using deuterium-acetone exch.......

Acknowledgements

We thank the Sanchez-Gurmaches and Wallace lab members for valuable discussions. This work was supported by grants from the American Heart Association (18CDA34080527 to JSG and 19POST34380545 to RM), the NIH (R21OD031907 to JSG), a CCHMC Trustee Award, a CCHMC Center for Pediatric Genomics Award, and a CCHMC Center for Mendelian Genomics & Therapeutics Award. This work was supported in part by NIH P30 DK078392 of the Digestive Diseases Research Core Center in Cincinnati. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. RT and MW were supported by a UCD Ad Astra Fello....

Materials

NameCompanyCatalog NumberComments
4 mL Glass VialsFisher Scientific14-955-334
0.2 µm filterOlympus Plastic25-244
26G needeled syringesBD309597
AcetoneMerck34850
AcetonitrileMerck900667
Blue GC screw cap with septaAgilent5190-1599
CentrifugeEppendorf5424R
ChloroformSigma366927
Deuterium oxideSigma151882
Di-tert-butyl-4-methylphenol (BHT)
Select FAME Column
MerckB1378
Di-tert-butyl-4-methylphenol (BHT)
Select FAME Column
AgilentCP7419
EDTA tubeSarstedt411395105
EthanolMerck51976
Hexadecenoic-d31 AcidLarodan71-1631
HexaneMerck34859
MethanolMerck34860
Microcentrifuge tubeOlympus Plastic24-282
Mouse environmental chamberCaronCaron 7001-33
Potasium ChlorideFisher BioreagentsBP366-500
Potasium PhosphateMP Biomedicals194727
SafeLock microcentrifuge tubesEppendorf30120086
Screw top amber GC vialAgilent5182-0716
Sodium ChlorideFisher BioreagentsBP358-212
Sodium HydroxideMerckS5881
Sodium Phosphate, dibasicFisher BioreagentsBP332-500
Sodium SulfateMerck239313
Sulfuric AcidMerck258105
Vial insertAgilent5183-2088

References

  1. . The Lancet, Diabetes Endocrinology. Childhood obesity: a growing pandemic. The Lancet. Diabetes & Endocrinology. 10 (1), 1 (2022).
  2. Gonzalez-Muniesa, P., et al. Obesity. Nature Reviews Disease Primers. 3, 17034 (2017).
  3. ....

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Keywords Fatty Acid SynthesisBrown Adipose TissueDeuterium OxideNon invasiveIsotope labeled GlucoseMetabolic StatusTissue DissectionTissue ExtractionChloroform methanol ExtractionFatty Acid MethylationGC MS Analysis

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