Name: quantitative determination of de novo fatty acid synthesis in brown adipose tissue using deuterium oxide
Uploaded: 2023-05-12T12:10:00
Description: Watch this Scientific Journal Video about Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide at JoVE.com

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 &#38; 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 Fellowship.

All experiments were approved by the Institutional Animal Care and Use Committee at Cincinnati Children&#39;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.
<ol>
	<li>For intraperitoneal injection: generate 0.9% w/v saline D2O by dissolving 9 g of NaCl per liter of D2O. Filter through a...

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...

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 simplicity of using portable glucose monitors4. Whole body glucose and lipid oxidation relative rates are also routinely estimated based on the respiratory exchange ratio from indirect calorimetry assays5,6. However, the majority of all other aspects of metabolism are not routinely functionally assessed. This leads to incomplete interpretations of the metabolic status and missed therapeutic options. One of the main such pathways is de novo lipogenesis.
De novo lipogenesis (DNL) is the process by which new fatty acids are generated from precursors. Glucose is considered to be the main precursor contributing to whole-body DNL7, however other precursors, such as acetate, fructose, lactate, and branched chain amino acids, have been shown to be relevant carbon sources in a spatial and condition dependent manner8,9,10,11,12. DNL is a key contributor to metabolic homeostasis and is essential for normal development13. Additionally, alterations in DNL have been associated with cancer14,15 and metabolic16,17,18 and cardiovascular diseases19,20.
The DNL pathway is composed of the core enzymatic components ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC1/2), and fatty acid synthase (FAS) which primarily produce palmitate, a 16-carbon saturated fatty acid. However, odd chain and branched chain fatty acids can also be produced at lower rates9. Elongases and desaturases further modify these fatty acids, creating a diverse range of fatty acids species useful for a variety of functions (e.g., long-term energy storage and manipulation of membrane fluidity).
The expression of the DNL enzymatic machinery is controlled by a short number of transcription factors. The most well described to date include the sterol regulatory element binding protein (SREBP) family, carbohydrate response element binding protein (ChREBP), and liver X receptor (LXR)21,22,23,24,25,26. Despite an apparent overlap in their functions, individual regulations based on cell type dominancy and physiological or pathological conditions have been reported21,22,27,28.
Remarkably, a number of inhibitors for selected steps of the DNL pathway have been approved for clinical use or are in the preclinical or clinical stages of development for a number of diseases, including obesity, nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH), and cardiovascular disease29. These efforts highlight the relevance of DNL in health and disease.
In recent years, the employment of methods to quantitatively assess de novo fatty acid synthesis has increased30. The most common method for assessing this is the use of heavy labelled water (D2O), where the heavy labelled hydrogen gets incorporated into acyl chains during synthesis both directly and indirectly, via deuterium exchange with the hydrogens of the DNL substrates NAPDH, acetyl-CoA, and malonyl-CoA. Although this approach is gaining in popularity, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we outline a method for quantitatively assessing the de novo synthesis of products of FAS using D2O and gas chromatography mass spectrometry (GCMS), with calculations previously developed by Lee et al.31. This method measures de novo fatty acid synthesis independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Here, we focus on the analysis of brown adipose tissue (BAT) due to its high levels of DNL and critical roles in maintaining metabolic homeostasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4 mL Glass Vials</td>
			<td>Fisher Scientific</td>
			<td>14-955-334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m filter</td>
			<td>Olympus Plastic</td>
			<td>25-244</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needeled syringes</td>
			<td>BD</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Merck</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Merck</td>
			<td>900667</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue GC screw cap with septa</td>
			<td>Agilent</td>
			<td>5190-1599</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Sigma</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl-4-methylphenol (BHT) 
			Select FAME Column</td>
			<td>Merck</td>
			<td>B1378</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl-4-methylphenol (BHT) 
			Select FAME Column</td>
			<td>Agilent</td>
			<td>CP7419</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA tube</td>
			<td>Sarstedt</td>
			<td>411395105</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>51976</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadecenoic-d31 Acid</td>
			<td>Larodan</td>
			<td>71-1631</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Merck</td>
			<td>34859</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Olympus Plastic</td>
			<td>24-282</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse environmental chamber</td>
			<td>Caron</td>
			<td>Caron 7001-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium Phosphate</td>
			<td>MP Biomedicals</td>
			<td>194727</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeLock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw top amber GC vial</td>
			<td>Agilent</td>
			<td>5182-0716</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate, dibasic</td>
			<td>Fisher Bioreagents</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Sulfate</td>
			<td>Merck</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Merck</td>
			<td>258105</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial insert</td>
			<td>Agilent</td>
			<td>5183-2088</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative determination of de novo fatty acid synthesis in brown adipose tissue using deuterium oxide

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.

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...

Watch this Scientific Journal Video about Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide at JoVE.com

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

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.

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic ...

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