Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach

Summary

Presented here is a protocol to measure in vivo adipose tissue kinetics in humans using the deuterium (²H)-labeling method.

Abstract

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (²H)-labeled water. The incorporation of ²H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Introduction

Obesity is a disease characterized by excess white adipose tissue (AT) and is a significant risk factor for the development of Type II diabetes and cardiovascular disease¹. White AT is a highly plastic organ that stores energy in the form of triacylglycerols (TGs) and is essential for metabolic homeostasis². White AT retains the ability to expand, reduce, and remodel during adulthood³, and the AT mass is determined by dynamic changes in the adipocyte volume (via TG synthesis and breakdown), continual adipocyte formation via the proliferation and differentiation of pre-adipocytes (i.e., hyperplasia or adipogenesis), and adipose cell death⁴.

Evidence suggests that there is an important link between the subcutaneous AT turnover (e.g., adipocyte formation and death) and cardiometabolic health^5,6,7,8, and the role of adipogenesis in the pathogenesis of obesity-related disorders remains debatable⁴. However, little is known about in vivo AT turnover in humans due, in part, to the slow turnover rate of the cellular components of the AT and the complexity of directly labeling their metabolic precursors in vivo. While in vitro methods have provided some insight, these approaches do not provide a comprehensive in vivo assessment within the natural milieu of the AT.

A method was developed by the Hellerstein laboratory⁹ to assess in vivo AT turnover using the incorporation of the stable isotope deuterium (²H) from heavy water (²H₂O) into the AT (Figure 1)¹⁰. The protocol, which has been validated in mice and humans, includes an initial ramp-up of ²H₂O to increase the ²H enrichment of the body water, followed by adequate daily intake to maintain stable, near-plateau enrichment values. The ²H from the ²H₂O (i.e., heavy water) is incorporated into the deoxyribose (dR) moiety of deoxyribonucleotides in the DNA of adipose cells, and the isotope enrichment is measured in the DNA via mass spectrometry and the application of mass isotopomer distribution analysis (MIDA)^9,10,11. Labeling the deoxyribose moiety of purine deoxyribonucleotides in DNA with stable isotope precursors has several advantages over previous methods, such as those that involved labeling with pyrimidine nucleotide base moieties (e.g., from ³H-thymidine or bromo-deoxyuridine). Of note, the endogenous reincorporation of bases, especially for pyrimidines, but not dR, in replicating DNA previously confounded the interpretation of label incorporation¹². In addition, the incorporation of a stable isotope label into dR causes no genotoxicity, in contrast to the incorporation of radioactive or genotoxic agents such as ³H (tritium) or bromo-deoxyuridine. Therefore, the long-term use of this technique in animal models and humans is safe.

The measurement of ²H-labeled DNA synthesis denotes the passage of a cell through the S-phase of cell division and identifies newly formed pre-adipocytes and adipocytes (via pre-adipocyte differentiation) or adipogenesis¹³. Cells that undergo rapid turnover (e.g., monocytes) replace their DNA quickly and reach a plateau in ²H-enrichment, thus providing an internal reference for the assay. The ratio of the ²H-enrichment of DNA from adipose cells to that of monocytes (reference cells) or the integrated body ²H₂O measurement allows the calculation of the fraction of newly synthesized adipose cells. Herein, this protocol describes methods to measure in vivo adipose cell turnover (adipogenesis) rates in humans via the ²H metabolic labeling protocol, including refined techniques to purify the adipocytes via negative immune selection and to enrich the pre-adipocyte population¹⁴.

Protocol

Pennington Biomedical Research Center's Institutional Review Board (IRB) approved all the procedures (#10039-PBRC), and all human subjects gave written informed consent.

1. Eight week ²H₂O-labeling period

1. Administer aliquots of either 70% or 99.9% deuterium-labeled water (²H₂O) in sterile plastic containers.
2. Instruct the participants to drink 35 mL doses of 99.9% enriched ²H₂O or 40 mL doses of 70% enriched ²H₂O three times per day in week 1 (priming period) and to drink two 35 mL or 50 mL doses, respectively, per day in weeks 2-8.
   NOTE: Transient dizziness or vertigo are the only known adverse effects of ²H₂O intake and are related to rapid changes in the bulk water flow, which are perceived by the hair follicles of the inner ear. Hence, instruct the participants to take the doses at least ~2 h apart to avoid the rare occurrence of transient dizziness or vertigo¹⁵. For the same reason, do not make up for missed doses by doubling a single dose. Administering the dose as described above (separated by ≥2 h) makes this adverse effect extremely rare in human subjects (<1% of several hundred subjects).
3. Monitor the compliance with ²H₂O intake through urine collections to measure the ²H enrichment in the body water and also by the weekly return of the empty vials for counting.
   1. Clean the urine samples using activated charcoal and a filter.
      1. Add 8 mL of urine to a 10 mL tube with 1 mL of activated charcoal.
      2. Place the sample on a rocker for 10 min, and centrifuge for 5 min at 800 x g so that the charcoal moves to the bottom of the tube. Filter the sample with a 0.2 µm syringe filter.
   2. Measure the ²H₂O enrichment in the body water (urine) by isotope ratio mass spectrometry (IRMS).
      NOTE: ²H₂O enrichment can be analyzed by different methods, including gas chromatography-mass spectrometry¹⁶, high-temperature conversion elemental analyzer (TC/EA) coupled with IRMS¹⁷, cavity ring-down infrared spectroscopy (IRIS)¹⁸, or with an H/Device attached to a mass spectrometer (e.g., IRMS)¹⁹.
   3. Use the mean ²H₂O enrichments measured in the urine during the 8 week labeling period to calculate the precursor ²H₂O exposure and the fraction of newly synthesized AT cells (see section 7 and section 8 below).

NOTE: The ²H₂O labeling protocol maintains near-plateau ²H enrichment in the body water within the range of 1.0%-2.5% for the duration of the 8 week labeling period (Figure 2)¹⁰.​

2. Adipose tissue biopsy collections from human subjects

1. After cleansing the skin with povidone-iodine solution, administer topical anesthesia (e.g., 2% lidocaine/0.5% bupivacaine), make a ~0.75 cm incision in the skin, and collect subcutaneous AT biopsies via the needle lipoaspiration technique under sterile conditions¹³.
2. Weigh a sterile 50 mL tube containing 5 mL of room temperature (RT) 1 M HEPES buffer, pH 7.3. Immediately place the AT in the tube with the HEPES buffer for processing.
3. Weigh the 50 mL tube containing the AT, and record the weight.

3. Isolation of purified adipocytes

1. Add a type 1 collagenase /HEPES (2 mg/mL) solution to 2 g/mL AT.
2. Digest the AT by incubating in a water bath with shaking (100 rpm) for 1 h at 37 °C until there is a homogenous mixture with a few large, intact pieces of AT.
3. Centrifuge the tube at 500 x g for 8 min at RT to separate the adipocytes and the stromal-vascular fraction (SVF).
4. Gently remove the top fat layer (adipocytes), and transfer it to a separate tube. Do not disrupt the pellet (SVF) at the bottom of the tube.
5. As previously described¹⁴, purify the adipocytes using immunomagnetic cell separation.
   NOTE: This important step is done to "clean" the adipocytes, as other cell types with fast cell turnover, including hematopoietic, endothelial, and stem cells, may adhere to the floating adipocytes and impact the measurements.
   1. Place ~400 µL of adipocytes into a 5 mL tube for negative immuno-purification.
   2. Add FcR blocking antibody (100 µL/mL).
   3. Add a cocktail of biotinylated antibodies against markers of endothelial cells (anti-human CD31; 1:100), hematopoietic cells (anti-human CD45; 1:400), and mesenchymal stem cells (anti-human CD34; 1:100) to the adipocyte solution for 15 min at RT.
   4. Add a biotin selection cocktail (100 µL/mL) to the adipocyte solution, mix well, and incubate for 15 min at RT with gentle tilting/rotation.
   5. Mix the magnetic nanoparticles by pipetting up and down, add the particles (50 µL/mL) to the adipocyte solution, mix well, and incubate for 10 min at RT with gentle rotation (10 rpm).
   6. Add PBS/2% FBS/1 mM EDTA buffer to the adipocyte solution for a total volume of 1 mL.
   7. Place the tube in the magnet, and let it sit for 5 min.
   8. Pick up the magnet, and in one continuous motion, invert the magnet containing the tube, and pour the contents into a new 5 mL tube. Do not tap the tube while pouring. The cells attached to the antibodies are bound by the magnetic nanoparticles and removed, while the immuno-purified adipocytes are retained.
   9. Flash-freeze the purified adipocytes in liquid N₂, and store them at −80 °C until the DNA extraction.

4. Isolation of pre-adipocytes

1. To isolate an enriched population of pre-adipocytes, employ a protocol to exploit their ability to attach to plastic after a short-term culture of the SVF²⁰.
2. In a laminar flow hood, resuspend the SVF pellet in 5 mL of erythrocyte lysis buffer for 5-10 min at RT (mix thoroughly), and centrifuge at 800 x g. Remove the supernatant, and resuspend the pellet in 10% FBS in alpha (α)MEM.
3. Plate the cells on a plastic culture dish for ~8-12 h in a tissue culture incubator at 37 °C and with a 5% CO₂ atmosphere.
4. After ~8-12 h, gently wash the non-adherent cells from the culture plate with PBS inside a laminar flow hood.
5. After aspirating the PBS, add 1-1.5 mL of 0.25% trypsin/1 mM EDTA to the culture dish to detach the adherent cells (enriched population of pre-adipocytes). Place the culture dish in the incubator at 37 °C for ~5-8 min to help lift the cells.
6. Add 10% FBS/αMEM to the plate, and wash the plate thoroughly to collect all the cells from the plate. Transfer the cell solution into a 15 mL or 50 mL tube, and centrifuge at 800 x g for 8 min.
7. Remove the supernatant, and store the pellet (pre-adipocytes) at −80 °C until the DNA extraction.

5. Isolation of blood monocytes

NOTE: The monocytes are analyzed to represent a (nearly) completely turned-over cell population, and the measurement of the ²H enrichment in the monocytes can be used as a reference marker of ²H₂O exposure in each individual. Alternatively, the body ²H₂O enrichment can be measured and used to calculate the ²H₂O exposure.

1. Draw fresh, whole blood from human subjects into vacutainer tubes containing EDTA.
2. Centrifuge the blood at 1,000-2,000 x g for 15 min at 4 °C.
   NOTE: Do not use the brake.
3. Remove the top layer of plasma. Be careful not to touch the white buffy coat just under the plasma.
4. Aspirate the white buffy coat using a transfer pipette, and transfer it to a 50 mL tube. Add ~10 mL of PBS to the buffy coat.
5. Gently add 10 mL of a density gradient medium to layer the buffy coat.
   NOTE: Make sure the tip of the pipette touches the bottom of the tube, and eject carefully so that there will be a clear layer of density gradient medium at the bottom. Avoid introducing bubbles.
6. Centrifuge the tube at 800 x g for 30 min with the brake off.
7. Pipette out (~10 mL) the white layer (mononuclear fraction), making circular movements with the tip of the transfer pipette close to the border but not touching the upper layer. Transfer to a new 50 mL tube, and add 10 mL of PBS. Centrifuge at 800 x g for 5 min (do not use the brake), and discard the supernatant.
8. Add 5 mL of erythrocyte lysis buffer to the pellet. Mix, and let it sit for 2 min at RT.
9. Add 5 mL of PBS with 0.1% BSA to the erythrocyte lysis buffer/pellet solution, centrifuge at 800 x g for 5 min, and discard the supernatant.
10. Isolate the monocytes as CD14⁺ cells using immuno-magnetic beads. Perform the isolation of the CD14⁺ cells by following the manufacturer's protocol.
11. Store the isolated monocytes at −80 °C until the DNA extraction.

6. DNA preparation (isolation, hydrolysis, and derivatization)

1. Isolate the DNA from the pre-adipocytes, adipocytes, and blood monocytes using a DNA extraction kit following the manufacturer's instructions.
2. To free the deoxyribonucleosides, enzymatically hydrolyze the DNA (~200 µL) overnight (no more than 24 h) at 37 °C in 50 µL of enzyme hydrolysis cocktail containing 1 mL of S1 nuclease, 1 mL of phosphatase enzyme, and 36.8 mL of 5x hydrolysis buffer in 16 mm x 100 mm (10 mL) screw-capped glass tubes. Additionally, include the following samples: a water blank, a hydrolysis cocktail blank, a column blank from the DNA extraction kit, and 500 ng of DNA standards.
   1. To prepare the 5x hydrolysis buffer, place 94 mL of molecular biology-grade pure water into a sterile beaker. Weigh out 3.08 g of sodium acetate and 21.5 mg of zinc sulfate, add water, and mix until dissolved. Adjust the pH to 5.0 with glacial acetic acid, and test using pH paper. Add water to reach a total volume of 100 mL.
   2. To prepare the phosphatase enzyme, resuspend a vial of acid phosphatase in 1 mL of pure water.
   3. To prepare the 0.5 U/µL S1 nuclease enzyme, dilute 2.5 µL of S1 nuclease into 2 mL of 1x hydrolysis buffer (add 1 mL of 5x hydrolysis buffer to 4 mL of pure water).
3. Derivatize the hydrolysates to pentafluorobenzylhydroxylamine (PFBHA) derivatives. Specifically, add the following directly to the digested DNA samples, including the standards and blanks, in 16 mm x 100 mm (10 mL) screw cap tubes: 100 µL of pentafluorobenzyl hydroxylamine hydrochloride (PFBHA; 1 mg/mL) and 75 µL of glacial acetic acid. Vortex (briefly), cap the vials, and place the hydrolysate samples on a heating block set at 100 °C for 30 min.
4. Remove the samples, and let them cool for ~5 min to RT. After cooling, add 2 mL of acetic anhydride and 100 µL of 1-methylimidazole to each tube under a fume hood. Set the tubes on a 100 °C heat block for 5 min.
5. Remove the samples, and then allow them to cool for ~15-20 min. Once cool, add 3 mL of molecular biology-grade water to each sample, vortex briefly, and let them sit for 10 min.
6. Add 2 mL of dichloromethane (DCM) to the tubes, and vortex the samples vigorously for 15 s to extract the derivative into the organic phase.
7. Centrifuge at 800 x g for 5 min to separate the phases. Carefully transfer the bottom dichloromethane layer into a clean 1.6 mL GC vial.
   NOTE: Do not transfer any of the aqueous phase, as this will increase the background.
8. Evaporate to dryness with nitrogen for ~20 min to remove the DCM and residual acetic acid, followed by a ~10 min dry in a speed vacuum at RT to remove all traces of the reagents.

7. Gas chromatography-mass spectrometry (GC-MS) analysis of the DNA

1. Once dry, resuspend the PFBHA derivatives in 150 µL of ethyl acetate, and cap. Next, analyze for the incorporation of ²H into the DNA on a gas chromatography (GC)/mass spectrometry (MS) instrument equipped with a DB-225 column using methane negative chemical ionization and collecting the ions in selective ion-monitoring mode at m/z 435, m/z 436, and m/z 437 (representing the M₀, M₁, and M₂ mass isotopomers, respectively). Follow the manufacturer's instructions for the instrument used.
   1. Set the GC conditions as follows: column: 30 m x 0.25 mm ID x 0.25 µm film coating; chromatograph: helium carrier gas at 1.0 mL/min constant flow; pulsed split-less injections; injector temperature: 250 °C; MS transfer line: 325 °C; oven conditions: initial temperature 100 °C, hold 2 min, ramp at 40 °C/min to a temperature of 220 °C, hold 7.5 min, second ramp at 40 °C/min to 320 °C, hold for 1 min. The total runtime is 16.0 min. The typical dR elution times are 10.5 min and 10.8 min for the first and second peaks, respectively.
   2. Set the MS conditions as follows: methane negative chemical ionization source with detection in single ion monitoring mode; ions collected for dR: m/z 435, m/z 436, and m/z 437 (representing M₀, M₁, and M₂ mass isotopomers, respectively).
      NOTE: Siliconize the open glass liner with a small plug of siliconized glass wool in the lower portion of the liner. Change this liner often (every ~100 injections).
2. Calculate the mass isotopomer abundance M1 ratios in the unenriched (natural abundance) dR derivative as follows:
   M1 ratio = M1 abundance/(sum of M0, M1, and M2 abundances)
   Then, subtract the natural abundance M1 ratios from the M1 ratios in the dR of the labeled samples to calculate the enrichments (excess isotope abundances, or %EM1)²¹.
3. Measure the mass isotopomer abundances of the baseline (unenriched) DNA standards concurrently over a range of M0 ion abundances that span the range of M0 ion abundances in the samples analyzed.
   NOTE: This step is essential to correct for the "abundance sensitivity" of isotope ratios measured by GC/MS²².
4. Construct a graph of the measured M0 ion abundance versus the measured mass isotopomer M1 ratios in the unlabeled standards to determine the on-column sample concentration that is most accurate for the M1 mass isotopomer (i.e., where it is closest to the known, calculated natural abundance M1 ratio, or the "sweet spot"). The M1 ratio for this unenriched dR derivative is 0.1669.
5. Inject the experimental samples so that they are as close as possible in the M0 peak area to this "sweet spot". Use a quadratic regression of the M1 ratio of the unenriched standards versus the M0 peak area to estimate an "adjusted natural abundance" M1 ratio for each sample at its particular M0 concentration. Subtract this from the adipose cell sample M1 ratio to yield the percent enrichment above the natural abundance, or %EM1 (Figure 3)²³.
6. Calculate the theoretical maximum M₁ enrichment (EM1*) in the adipose cells using mass isotopomer distribution analysis (MIDA) equations²⁴ based on the body ²H₂O exposure (measured in the urine) integrated over the 8 week period (Figure 4) or the interim measured monocyte enrichments (step 7.5).

8. Calculation of the fraction of newly synthesized cells, or in vivo adipogenesis

1. Calculate the fraction of new cells (%), which is a measure of the in vivo adipogenesis or the formation of newly synthesized pre-adipocytes or adipocytes, using the following formula:
   Fraction of new cells (%) = [(M₁ enrichment in the sample [adipose] cells)/EM1* (theoretical maximum M₁ enrichment)] x 100
   NOTE: *The ²H enrichment in monocytes can also be used as the denominator of the equation. This measurement in monocytes represents a reference cell marker of the ²H₂O exposure in each individual and may be used to confirm the calculations of theoretical maximum M₁enrichment from the measured body ²H₂O values.

Results

The ²H₂O labeling protocol (section 1) maintains near-plateau ²H enrichment in the body water within the range of 1.0%-2.5% for the duration of the 8 week labeling period¹⁰, as shown in Figure 2. A previous study utilized the ²H₂O labeling protocol to assess adipose kinetics via the incorporation of ²H into the DNA of adipose cells, as detailed in sections 2-8, and reported that in vivo

Discussion

In vivo assessments are necessary to provide new knowledge on the dynamics of white AT turnover and its role in obesity and related metabolic diseases, as in vitro assessments do not encompass the natural environment of the AT. Although the use of retrospective radiocarbon dating to assess adipose dynamics has been informative^7,25, this approach is not suitable for capturing dynamic changes during prospective intervention studies. The ²

Materials

Name	Company	Catalog Number	Comments
1-methylimidazole	MilliporeSigma	336092
2H2O	Sigma Aldrich
Acetic anhydride	Aldridge	539996
ACK Lysing Buffer (erythrocyte lysis buffer)	Quality Biological Inc (VWR)	10128-802
Agilent 6890/5973 GC/MS	Agilent
Anti-human CD31 (PECAM-1) Biotin	Invitrogen	13-0319-82
Anti-human CD34 Biotin	Invitrogen	13-0349-82
Anti-human CD45	BioLegend	304004
Antibiotic Antimycotic Solution	MilliporeSigma	A5955
Collagenase type 1	Worthington Biochemical Corporation	LS004196
Deoxyribose (2-deoxy d-ribose)	MilliporeSigma	31170
Deuterium Oxide	MilliporeSigma	756822
DB-225 column (30m, 0.25mm, 0.25um)	J&W Scientific	122-2232
Dichloromethane (DCM)	MilliporeSigma	34856
DNA standard (calf thymus DNA)	MilliporeSigma	D4764
Dneasy Blood and Tissue Kit (DNA extraction kit)	Qiagen	69504
Easy Sep Human Biotin kit	Stem Cell Technologies	17663
EasySep Human CD14 Positive Selection Cocktail	Stem Cell Technologies	18058C
Ethyl acetate	Fisher	EX0241-1
Falcon 5 mL Round Bottom Polystyrene Test Tube	VWR	60819-295
Ficoll-Paque Plus	MilliporeSigma	GE17-1440-02
GC vials (2 mL)	Fisher	C-4011-1W
GC vial inserts	Fisher	C-4011-631; C-4012-530
Glacial acetic acid	Fisher	AC14893-0010
Glass tubes (for hydrolysis)	Fisher	14-959-35AA
HEPES buffer	ThermoFisher	15630080
Hyclone Water, molecular biology grade	Thomas Scientific	SH30538.02
MEM alpha	Fisher Scientific	32561-037
PFBHA (o-(2, 3, 4, 5, 6)-penatfluorobenzylhydroxylamin hydrochloride)	MilliporeSigma	194484
pH indicator strips	Fisher	987618
Phosphatase acid	Calbiochem (VWR)	80602-592
S1 nuclease (from Aspergillus oryzae)	MilliporeSigma	N5661
Sodium sulfate	MilliporeSigma	23913