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 ...

Contrary to other metabolic pathways like glucose disposal, fatty acid synthesis is not routinely assessed, leading to incomplete interpretations of the metabolic status. The key benefits of this method is that it's relatively non-invasive, and it's likely a better reflection of fatty acid synthesis flux than using isotope-labeled glucose where changes in upstream glucose handling can impact results. Here, we focus on brown adipose tissue, but this method is potentially useful to study any tissue in any mouse model.To begin, place the samples on dry ice. Take a pre-labeled microcentrifuge tube, place it on an analytical balance and tare the balance. Then place tweezers and a scalpel or steel razor blade on dry ice for 10 to 20 seconds to cool.Use the tweezers to take the frozen tissue sample out of the tube and place it on a plastic weight boat, which can be positioned on a flat block of dry ice or another pre-cooled surface. Using the scalpel or steel razor blade, dissect a small portion of the tissue equivalent to 5 to 15 milligrams in weight, and place it in the microcentrifuge tube. Record the exact weight and repeat for each specimen.Add one microliter per milligram of 10 millimolar hexadecanoic D31 acid, followed by three five millimeter stainless steel beads to each sample. Then add 250 microliters of methanol, 250 microliters of water, and 500 microliters of chloroform to each sample with beads. Place the tubes in a pre-cooled block of a grinding mill and mix the samples at a vibrational frequency of 25 hertz for five minutes.Remove the beads using a magnet and centrifuge the samples at 12, 000 G for 10 minutes at four degrees Celsius. Using a micropipette, take a fixed volume of the bottom phase of each sample into correspondingly labeled microcentrifuge tubes. Add 500 microliters of chloroform to the remaining sample and repeat these steps.Place the tubes with the combined lower phase under nitrogen gas or in a chloroform-resistant refrigerated centrifugal vacuum at four degrees Celsius until completely dry. Pipette 98 milliliters of anhydrous methanol into a glass media bottle. And slowly add two milliliters of anhydrous sulfuric acid in the fume hood to make 2%sulfuric acid in methanol.Mix by swirling the closed bottle. Add 500 microliters of this 2%sulfuric acid in methanol solution to each sample and vortex briefly. Incubate the samples on the heat block at 50 degrees Celsius for two hours and add 100 microliters of saturated sodium chloride solution and 500 microliters of hexane to each sample.Vortex the samples vigorously at room temperature for one minute and leave the samples to sit for one minute. The two phases should be apparent after this. Collect the upper phase into a fresh microcentrifuge tube.Then repeat the addition of hexane. And after phase separation, collect the second upper phase samples into the same labeled tubes. Dry the samples at room temperature under nitrogen gas and resuspend the samples in 20 microliters of hexane per milligram of original tissue weight.Transfer the samples immediately to a glass gas chromatography vial with a glass insert. Inject the samples into a single quadruple gas chromatography mass spectrometer to determine the abundance of fatty acid methyl esters or FAMEs isotopologues. Inject one microliter of sample into a split or splitless inlet at an inlet temperature of 270 degrees Celsius using helium as the carrier gas.In labeled safe-lock microcentrifuge tubes, combine 10 microliters of each plasma sample or standard four microliters of 10 molar sodium hydroxide and four microliters of 5%acetone in acetonitrile. Perform this in triplicate for each sample. After incubating the samples overnight at room temperature, add 450 to 550 milligrams of sodium sulfate to each sample, add 600 microliters of chloroform to each tube in the fume hood, and vigorously vortex for 15 seconds.Centrifuge the samples at 300 G for two minutes. Take labeled glass GCMS vials with glass inserts and transfer the triplicate and then 80 microliter aliquots of the supernatant from each sample to the vials. Cap the vials tightly for the GCMS analysis.The percentage of D2O enrichment in the plasma of mice over multiple time points is shown here. It was found that body water is enriched in the range of 2.5 to 6%that a baseline level of deuterium enrichment in body water is rapidly achieved in one hour and maintained for the duration of the study. The mass isotopologue distribution of palmitate in brown adipose tissue after three days of D2O administration at room temperature and thermoneutrality is shown here.This result shows a higher M1 and M2 deuterium enrichment at room temperature. Brown adipose tissue molar enrichment and plasma molar enrichment of a range of fatty acids after three days of D2O administration at room temperature and thermoneutrality are presented in this figure. The colder temperature enrichment was found in a broad range of fatty acids in brown adipose tissue.Notably, plasma total fatty acid enrichment does not follow the same trend as brown adipose tissue, but instead, fatty acid enrichment is increased with thermoneutrality. These images represent the total abundance and the de novo synthesized palmitate in brown adipose tissue after three days of D2O administration. The result shows an increase in the total palmitate synthesis at room temperature.Be careful when running fatty acids on the GCMS. Make sure palmitate is not overloaded as just can give artificially high isotope enrichment values. Additionally, when analyzing acetone on the GCMS, make sure there is no background acetone peak before running the samples, so run blanks to remove any acetone in the system.This method looks at total fatty acids across multiple lipid classes, but if you want to understand how fatty acid synthesis is impacting specific lipid classes, you can first separate these lipid classes using chromatography and then extract fatty acids from these fractions. Because lipogenesis is a key contributor to normal development, but also to many diseases, this protocol can be useful in many fields.

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

Abstract