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

요약

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.

초록

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.

서문

Obesity and associated metabolic diseases are a pandemic that endanger present and future generations^1,2. 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 assays^5,6. 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 manner^8,9,10,11,12. DNL is a key contributor to metabolic homeostasis and is essential for normal development¹³. Additionally, alterations in DNL have been associated with cancer^14,15 and metabolic^16,17,18 and cardiovascular diseases^19,20.

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 reported^21,22,27,28.

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.

프로토콜

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 non-pyrogenic 0.2 µm filter to sterilize.
2. For drinking D₂O-water: generate 8% v/v D₂O-enriched water to be used as drinking water by mixing 80 mL of D₂O per 920 mL of regular drinking water. Regular drinking water can be obtained from the mouse facility. Filter through a non-pyrogenic 0.2 µm filter to sterilize.

2. Modulation of BAT activity by temperature acclimation

1. Separate the mice so there are two mice per cage 2 weeks prior to the start of temperature acclimation. Change the water supply to water bottles for the mice to adapt to and maintain all the cages at 22 °C.
2. Prepare the mouse environmental chambers 1 week before starting the temperature acclimation by setting the appropriate temperatures: 30 °C for thermoneutrality, 22 °C for room temperature, and 18 °C for cold exposure.
3. At the start of temperature acclimation, replace the cages with new ones with no environmental enrichment (to avoid nests). Move the cages to their respective temperatures.
   NOTE: Cages assigned to thermoneutrality will stay at 30 °C for 4 weeks. Cages assigned to room temperature will stay at 22 °C for 4 weeks. Cages assigned to cold exposure will become progressively colder on a weekly schedule: 18 °C for the first week, 14 °C for the second week, 10 °C for the third week, and 6 °C for the fourth week.
4. Change soiled cages weekly for all conditions. Additionally, replace food and water bottles with new ones preadapted at the appropriate temperature for at least 24 h.
   NOTE: Be mindful about the food amounts added every week, especially for mice in the cold, since they will consume a significantly greater amount of food compared to normal conditions. Provide food ad libitum.

3. Administration of D​₂O

1. Inject each animal with 0.9% w/v saline-D₂O at 35 µL/g of body weight, 12 h-3 days before tissue collection, via intraperitoneal injection using a 1 mL syringe and 26 G needle.
   NOTE: Please see the discussion section for more information on selecting an appropriate labelling time.
2. Change the water bottles to bottles containing 8% v/v D₂O-enriched drinking water.

4. Plasma and tissue collection, processing, and storage

1. At the end of the experiment, sacrifice the mice using approved methodologies (e.g., carbon dioxide overdose followed by cervical dislocation).
   1. Use heat/cool pads or other methods to avoid sudden temperature changes before euthanasia that may affect the results. Euthanize the mice following approved methodologies. Proceed immediately to blood and tissue collection.
2. Collect blood through cardiac puncture using a 26 G needle and store in an ethylenediaminetetraacetic acid blood collection tube. Keep the blood on ice until further processing.
3. Cut open the skin along the middle line of the back of the mouse from the lower area of the thoracic cavity up to the upper area of the neck, while pulling the skin up to avoid affecting tissues below the skin. The interscapular BAT is located between the shoulder blades under a thin layer of white adipose tissue, and it is composed of two pyramidal shaped lobes.
   1. Clean the tissue by washing in ice cold phosphate buffered saline (PBS). Dab on a paper towel to eliminate excess liquid, weigh on an analytical scale, and collect in a microtube.
   2. Immediately flash freeze the tissue using liquid nitrogen. Other BAT depots can also be collected.
4. Centrifuge the blood samples at 10,000 x g for 10 min at 4 °C. After centrifugation, carefully collect the plasma without disturbing the red blood cell pellet. Transfer it to a new ice-cold microtube and flash freeze in liquid nitrogen.
5. Store brown fat and plasma samples at -80 °C until use.

5. Lipid extraction from adipose tissue

1. Before beginning the extraction
   1. Prepare a 1 mM solution of hexadecenoic-d₃₁ acid in methanol in a glass vial. This will serve as the internal fatty acid standard.
   2. Pre-cool the required amount of chloroform (CHCl₃) and methanol (CH₃OH) in a -80 °C freezer, or on dry ice.
      NOTE: The antioxidant di-tert-butyl-4-methylphenol (BHT) may be added to the CHCl₃ at a concentration of 0.01% w/v (2.5 mg/25 mL) to prevent oxidation of the double bonds in unsaturated fatty acids.
   3. Pre-label microcentrifuge tubes for each tissue sample and an extra tube to be used for a blank extraction.
      CAUTION: CH₃OH and CHCl₃ are highly volatile and toxic if inhaled. Use only in fume hoods.
      NOTE: Different brands of microcentrifuge tubes have differing levels of background palmitate and capabilities in terms of preventing solvent leakage. We recommend that a variety of tubes be tested first, to ensure that solvent leakage is prevented and that the tubes have minimal levels of contaminating palmitate. Please see Yao et al.³² for further discussion.
2. Take the samples out of the freezer and place on dry ice.
3. Place the pre-labelled microcentrifuge tube on an analytical balance and tare the balance. Place tweezers and a scalpel/steel razor blade on dry ice for 10-20 s to cool.
   1. Use the tweezers to take the frozen tissue sample out of the tube and place on a plastic weigh boat. The weigh boat may be placed on a flat block of dry ice or another pre-cooled surface.
   2. Using the scalpel or steel razor blade, dissect a small portion of the tissue, equivalent to 5-15 mg in weight. Place in the microcentrifuge tube and record the exact weight. Repeat for each specimen. Samples can be stored in the freezer at this point or can be advanced to the steps below for lipid extraction.
      NOTE: Ensure that the scalpel is properly cleaned with 70% ethanol between samples and a fresh weigh boat is used between each sample.
4. Add 1 µL/mg of 10 mM hexadecenoic-d₃₁(C16:0-d₃₁) acid to each sample.
   CAUTION: The following steps (5.4 to 5.8) should be carried out under a fume hood due to the inhalation risk of the solvents.
5. Add 250 µL of CH₃OH, 250 µL of H₂O, and 500 µL of CHCl₃ to each sample with three 5 mm stainless steel beads. Place the tubes in a pre-cooled block of a grinding mill and mix the samples at a vibrational frequency of 25 Hz for 5 min, or use guidelines recommended by the manufacturer for tissue samples. Remove the beads using a magnet.
6. Centrifuge the samples at 12,000 x g for 10 min at 4 °C.
   NOTE: After centrifugation, a clear biphasic separation should be observed with the upper aqueous phase containing polar metabolites and the lower organic phase containing lipids and fatty acids. If no separation is seen, add 250 µL of H₂O and repeat the vortex and centrifugation steps.
7. Using a micropipette, take a fixed volume from the bottom phase of each sample into correspondingly labelled microcentrifuge tubes.
8. Add 500 µL of CHCl₃ to the remaining sample and repeat steps 5.6-5.7.
9. Place the samples under nitrogen gas or in a CHCl₃-resistant refrigerated centrifugal vacuum at 4 °C until completely dry. Dried samples can be stored at -20 °C until ready for derivatization.
   NOTE: The top layer of each sample can also be collected at this point and dried as in step 5.9 in order to analyze polar metabolites.

6. Preparation of fatty acid methyl esters (FAMEs) and GCMS analysis

1. Acid-catalyzed esterification and transesterification to prepare FAMEs
   CAUTION: The following steps should be carried out under a fume hood due to the inhalation risk of the solvents.
   1. If the samples have been stored in freezer, dry under nitrogen for 5 min to ensure that no water is present.
   2. Using MS-grade solvents, pipette 98 mL of anhydrous CH₃OH into a glass media bottle. Slowly add 2 mL of anhydrous sulfuric acid in the fume hood to make 2% H₂SO₄ in CH₃OH. Mix by swirling the closed bottle.
   3. Add 500 µL of 2% H₂SO₄ in CH₃OH solution to each sample and vortex briefly.
   4. Incubate the samples on a heat block at 50 °C for 2 h.
   5. Remove the samples from the heat block and add 100 µL of saturated NaCl solution and 500 µL of hexane to each sample.
   6. Vortex the samples vigorously at room temperature for 1 min. Leave the samples to sit for 1 min; two phases should be apparent after this.
   7. Collect the upper phase into a fresh microcentrifuge tube (see note in section 5.1.3 for appropriate selection of the microcentrifuge tube).
   8. To maximize the yield, repeat steps 6.1.5-6.1.7, collecting the second sample into the same labelled tubes.
   9. Dry the samples at room temperature under nitrogen gas.
   10. Resuspend the samples in 20 µL/mg of hexane, relative to the original tissue weight, and transfer immediately to glass GC vial with a glass insert.
       NOTE: Work quickly while transferring samples to minimize evaporation.
2. GCMS analysis
   1. To determine the abundance of FAME isotopologues, inject the samples on a single quadrupole gas chromatography mass spectrometer (GCMS).
      NOTE: While many column types can be used to detect palmitate, the following temperature program was established for a GCMS column that has been developed for the separation of fatty acid cis/trans isomers, as detailed in the Table of Materials. This column has a length of 50 m with an 0.25 mm inner diameter.
   2. Inject 1 µL of sample in a split/splitless inlet at an inlet temperature of 270 °C, using helium as the carrier gas, flowing at 1 mL/min. Use a splitless injection for low abundant fatty acids with a total flow of 19 mL/min, a septum purge of 3 mL/min, and a purge flow to split vent of 15 mL/min at 0.75 min. Use a split ratio of 10:1-40:1 for high abundant fatty acids such as palmitate and oleate.
   3. Use the following oven parameters: an initial temperature of 80 °C; increase by 20 °C/min increments to 170 °C; increase by 1 °C/min increments to 204 °C; increase by 20 °C/min increments to 250 °C; and then hold at 250 °C for 10 min.
   4. Use the following mass selective detector (MSD) parameters: an electron impact ionization mode of 70 eV and scan over the range of 50-400 m/z with a scan speed of 1,562 (u/s) and a frequency of 4.1 scans/s. Use a transfer line at 280 °C, an ion source at 230 °C, and a quadrupole at 150 °C.
      ​NOTE: Other columns may be used, but the temperature program will vary.
   5. Sample sequence: Randomize the sample order of injection and inject at least two or three hexane blanks at the beginning of the sequence, after every fifth sample within the sequence, and two or three at the end of the sequence.
   6. Use instrument specific software, or free open-access software such as Metabolite-Detector³³, to integrate the ions in Table 1 for each fatty acid methyl ester.
      NOTE: We have outlined ions that cover M1-M5 isotopologues in Table 1 that we have found covers the amount of deuterium incorporation with this labelling window. However, this may need to be expanded if de novo flux is significantly higher and/or a longer labelling time is used.
   7. Use the abundance of each integrated ion to generate a mass isotopomer distribution, where the ion intensities can be converted to fractional abundance so that the sum of the mass isotopomer distribution equals one. Please see the example spreadsheet in the Supplementary File.
      NOTE: In order to accurately determine deuterium incorporation, correction of the natural isotope abundance then has to be employed to allow for the presence of naturally occurring isotopes such as ¹³C, ¹⁵N, and ²H. This is performed by applying a correction matrix as outlined by Fernandez et al.^34,35, and cannot be performed by subtracting the MID of an unlabelled measured metabolite from a labelled metabolite. In practice, we recommend the use of freely available software such as fluxfix³⁶, polyMID³⁷, or IsoCor³⁸ to turn raw data into fractional MIDs, and we have provided the formula for isotope correction for the methyl ester products of FAS in Table 1.
   8. To determine the quantity of palmitate present, utilize the following formula:
       
      where ion count refers to the sum of all palmitate isotopologues integrated in Table 1 and C16:0-d₃₁ refers to the internal standard hexadecenoic-d₃₁. The internal standard forms a separate peak to that of the endogenous palmitate. The relative abundance of other fatty acids may also be determined, but fatty acid specific isotope internal standards (with mass shifts greater than that observed from D₂O incorporation) or external standard curves may need to be employed for full quantitation. Use the blank extracted sample to determine the amount of background palmitate and subtract this from the final tissue value.
   9. Calculate the molar enrichment (ME) of palmitate by the following equation:
      
      ​where M_i is the normalized, fractional abundance of a palmitate isotopologue, and n is the number of possible palmitate isotopologues. For example, the ME of a palmitate molecule with the following fractional distribution, M1 = 0.25, M2 = 0.08, M3 = 0.02, is: (0.025*1) + (0.08*2) + (0.02*3) = 0.245 (See Supplementary File).

7. Deuterium acetone exchange of plasma samples to determine body water enrichment

1. Reaction
   1. Prepare 10 standards of deuterium in water, ranging from 0-9% v/v.
   2. Prepare a 5% v/v acetone/acetonitrile solution allowing for 4 µL per sample, including the standards from step 5.1.1.
   3. In labelled, safe-lock microcentrifuge tubes, combine 10 µL of each plasma sample or standard, 4 µL of 10 M NaOH, and 4 µL of 5% acetone/acetonitrile. Perform this in triplicate for each sample.
   4. Mix the samples gently by pipetting. Allow the samples to incubate at room temperature overnight.
2. Extraction
   1. After incubation, add 450-550 mg of Na₂SO₄ to each sample.
   2. In the fume hood, add 600 µL of CHCl₃ to each tube and vigorously vortex for 15 s.
   3. Centrifuge the samples at 300 x g for 2 min.
   4. Under a fume hood, transfer triplicate, 80 µL aliquots of the supernatant from each sample into labelled, glass GCMS vials with glass inserts and cap tightly.
3. GCMS analysis
   1. Separate samples on a column (30 m, 0.25 mm i.d, Agilent DB-35MS) and analyze on the attached mass spectrometer.
      1. Inject 1 µL of sample in a split/splitless inlet, with a split ratio of 40:1, a helium flow of 1 mL/min, and an inlet temperature of 270 °C.
      2. Use the following oven parameters: an initial temperature of 60 °C, increase by 20 °C/min increments to 100 °C, increase by 50 °C/min increments to 220 °C, and then hold at 220 °C for 1 min.
      3. Use the following mass selective detector (MSD) parameters: electron impact ionization mode at 70 eV with select ion monitoring of 58 and 59 m/z. Use a transfer line at 280 °C, an ion source at 230 °C, and a quadrupole at 150 °C.
         NOTE: Other low-bleed, bonded, crosslinked, mid-polarity columns may be used, but temperature program will vary.
   2. Set the liquid sampler method to begin with a CHCl₃ wash and then a randomized sequence of the samples, including additional CHCl₃ wash steps every six samples.
      NOTE: If acetone is used as a needle wash for the autosampler, replace with CHCl₃ or hexane and inject multiple blank CHCl₃ samples until any contaminating acetone peak is no longer evident in the chromatogram.
   3. Using this method, an acetone peak elutes at around 1.25 min. Integrate the data and calculate the fractional abundance of acetone enrichment following steps 6.2.6-6.2.7, with an adjustment to analyze peaks containing an m/z ratio of 58-60 as indicated in Table 1.
   4. Use the standards to generate a standard curve to determine the percentage of body water enrichment (p) of the test samples, based on the fractional enrichment of acetone.

8. In vivo de novo lipogenesis calculations

1. Calculate the fraction of newly synthesized fatty acids (FNS) that are direct products of FAS (i.e., palmitate, odd chain fatty acids, and mmBCFAs) in each specimen with the following equation:
   
   where ME is the average molar enrichment of a palmitate molecule (step 6.2.9), p is the deuterium enrichment in water from the corresponding plasma sample (step 7.3.4), and N is the number of exchangeable hydrogen atoms on palmitate where a deuterium can be incorporated.
2. Determine N using the equation below which was established by Lee et al.³¹:
   
3. Determine the molar amount of newly synthesized fatty acids (MNS) by:
   MNS = FNS x total fatty acid amount (nmol/mg tissue).
   NOTE: For example, if obtaining a palmitate molar enrichment (ME) of 0.245, a deuterium enrichment in body water (p) of 0.045, and a calculated N number of 22, the fractional synthesis of palmitate is 0.247. If the amount of palmitate present in the tissue is 2 mmol/mg, then the mmol of newly synthesized palmitate is 0.494 mmol/mg (see Supplementary File).

결과

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...

토론

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...

자료

Name	Company	Catalog Number	Comments
4 mL Glass Vials	Fisher Scientific	14-955-334
0.2 µm filter	Olympus Plastic	25-244
26G needeled syringes	BD	309597
Acetone	Merck	34850
Acetonitrile	Merck	900667
Blue GC screw cap with septa	Agilent	5190-1599
Centrifuge	Eppendorf	5424R
Chloroform	Sigma	366927
Deuterium oxide	Sigma	151882
Di-tert-butyl-4-methylphenol (BHT) Select FAME Column	Merck	B1378
Di-tert-butyl-4-methylphenol (BHT) Select FAME Column	Agilent	CP7419
EDTA tube	Sarstedt	411395105
Ethanol	Merck	51976
Hexadecenoic-d31 Acid	Larodan	71-1631
Hexane	Merck	34859
Methanol	Merck	34860
Microcentrifuge tube	Olympus Plastic	24-282
Mouse environmental chamber	Caron	Caron 7001-33
Potasium Chloride	Fisher Bioreagents	BP366-500
Potasium Phosphate	MP Biomedicals	194727
SafeLock microcentrifuge tubes	Eppendorf	30120086
Screw top amber GC vial	Agilent	5182-0716
Sodium Chloride	Fisher Bioreagents	BP358-212
Sodium Hydroxide	Merck	S5881
Sodium Phosphate, dibasic	Fisher Bioreagents	BP332-500
Sodium Sulfate	Merck	239313
Sulfuric Acid	Merck	258105
Vial insert	Agilent	5183-2088