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 ....

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.......

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