This study aimed to characterize bone marrow adipose tissue (BMAT) and determine if it expresses the brown fat marker UCP1. Three-dimensional electron microscopy revealed BMAT has an extensive mitochondrial network like brown fat. BMAT in young mice also appeared multilocular. However, unlike beige fat, BMAT volume was not depleted in UCP1-expressing cell death mice and the UCP1 reporter gene was not activated in BMAT. This suggests BMAT is a unique population between white and beige fat, with remodeling independent of UCP1. It likely specializes to support bone marrow functions through this UCP1-independent mechanism.

1. A
Bone marrow adipose tissue does not express UCP1
The role and regulatory mechanisms of white (WAT) and brown adipose tissues
(BAT) have been extensively explored. Previous research has also identified
adipocytes within the skeleton collectively known as bone marrow adipose tissue
(BMAT). These cells make up approximately 60-70% of bone marrow and 8% of
total fat mass in the average adult human. However, despite their prevalence, its
characterization as a white, brown, or beige adipocyte remains controversial.
Reconstruction of the bone marrow niche via three dimensional electron
microscopy (3D-EM) demonstrated that BMAT has an extensive mitochondrial
network interspersed around and between multiple lipid droplets. We also found
that BMAT adipocytes in 3-week old mice are multilocular and resemble brown
fat. Lastly, we found that a β3-adrenergic agonist CL316,243 was capable of
inducing lipid droplet remodeling in a subset of mouse bone marrow adipocytes in
vivo suggesting that BMAT has a distinctive phenotype which, like beige and
brown adipocytes, may undergo adrenergic-induced remodeling. To address this,
we developed two genetic mouse models. In the first, we used UCP1-Cre to drive
diphtheria toxin (DTA) expression and cell death (UCP1Cre+/DTA+).This allowed for
a quantitative, region-specific analysis of DTA-mediated UCP1 expression in
BMAT and WAT. In the second, UCP1-Cre mice were crossed with mTmG
reporter mice (UCP1Cre+/mTmG+) to allow visualization of UCP1 expressing cells in
bone and adipose tissues by monitoring GFP expression. We did not observe
BMAT depletion in the UCP1Cre+/DTA+ model of induced cell death following β3-
agonist stimulation. However, we discovered an exercise phenotype associated
with the UCP1-Cre mice that suggests a
remodeling of BMAT that is UCP1 independent.
Similarly, our reporter mouse model
demonstrated that UCP1-induced conversion of
the mTmG transgene did not occur in the
BMAT. Overall, our data suggests that BMAT
adipocytes, though capable of induced
remodeling, are not true UCP1-expressing beige
adipocytes. This supports the paradigm that
BMAT adipocytes are a unique subpopulation
which exists between white and beige cells, and,
based on our EM analyses, is likely specialized
to support cells within the skeletal niche in a
UCP1 independent manner.
1Department of Internal Medicine, Division of Bone and Mineral Diseases. Washington University, Saint Louis, MO, 63110, USA
2
Department of Internal Medicine, Division of Endocrinology, Metabolism and Lipid Research. Washington University, Saint Louis, MO, 63110, USA
References:
1. Robles H, Park S, Joens MS, Fitzpatrick JAJ, Craft CS, Scheller EL. Characterization of the bone
marrow adipocyte niche with three-dimensional electron microscopy. Bone. 2018
2. Scheller EL, Khandaker S, Learman BS, Cawthorn WP, Anderson LM, Pham HA, et al. Bone
marrow adipocytes resist lipolysis and remodeling in response to beta-adrenergic stimulation. Bone.
2018
Washington University Center for Cellular Imaging (WUCCI):
• Michael Shih, Matthew Joens, James Fitzpatrick
Funding:
• This project was supported National Institute of Health R00-DE024178, Washington University’s
Musculoskeletal Research Center award P30 AR057235, Diabetes Research Center Imaging
Grant P30 DK020579 and a grant from the Children’s Discovery Institute of Washington
University
Initially we observed that BMAT has beige/brown-like characteristics due to its expansive
mithochondrial network around and between its multiple lipid droplets, its multi-locular
appearance in adolescent mice, and its capacity for induced lipid remodeling. However,
unlike true beige adipocytes we failed to observe an increased loss of bone marrow fat
volume in the UCP1Cre+/DTA+ model of induced cell death even after treatment with a β3-
adrenergic agonist. We also did not observe activation of mTmG in the UCP1 reporter
mouse model in BMAT. However, we did demonstrate that UCP1Cre+/DTA+ is associated with
an exercise phenotype that likely affects depletion of BMAT. These findings suggest that
bone marrow adipocytes are not truly brown/beige, and are their own unique population that
is likely specialized to support functions within the skeletal niche that is UCP1 independent.
Future Direction of research includes…
• Studying the causes of BMAT deficiency in adult mice
• Defining the signaling pathways of marrow adipose tissues and their role in skeletal and
metabolic homeostasis
PERILIPIN CGRP
Figure 1a. Figure 1b.
(A) 2D image from a
FIB-SEM dataset
showing multiple
droplets budding off a
main adipocyte. (B)
The reconstruction
showed a large lipid
droplet and smaller
lipid droplets
enmeshed by a dense
mitochondrial network.
A B
Figure 1. (A) Representative histology of
WAT, BMAT, and BAT. (B) BMAT are
morphologically similar to WAT; however, it
is unclear where they fall on this ‘white-
brown-beige’ spectrum.
Prox
EpiphysisG
P
Tib/Fib
Junction
D
istalTibia
TotalTibia
0
20
40
60
MAT/MarrowVolume(%)
Control + Saline
Control + CL316
UCP1-Dta + Saline
UCP1-Dta + CL316
*
20
40
60
MAT/MarrowVolume(%)
Control + Saline
Control + CL316
UCP1-Dta + Saline
UCP1-Dta + CL316
*
20
40
60
MAT/MarrowVolume(%)
Control + Saline
Control + CL316
UCP1-Dta + Saline
UCP1-Dta + CL316
*
BB
A B
A
C
Hero Robles1, MR Lorenz1, ED Hilker1, KL Magee1, J Procknow1, Z Wang1, CA Harris2, EL Scheller1, CS Craft1
Figure 3. “Multilocularity” of BMAT
adipocytes in 3 week old mice. BMAT, WAT
and BAT were stained with antibodies against
perilipin to highlight adipocytes, DAPI to stain
nuclei and endomucin to stain vasculature.
Representative sections from the proximal and
distal areas of the tibia bone, 20x magnification
(scale bar = 100 µm), show that bone marrow
adipocytes in 3-week old mice (A,B) are multi-
locular. Sections of WAT (C) and BAT (D) are
provided as comparison. Imaging suggests
developing BMAT progresses through a multi-
locular phase before consolidating into larger,
unilocular adipocytes. In some cases, this
resembles BAT. Solid white boxes show a
magnified inset of each fat tissue type.
Figure 5. 3D micro computed tomography
reconstructions of bone marrow adipose
tissue overlaid on bone (A) and region-
specific BMAT quantification (B)
demonstrate a decrease in BMAT percent
volume in control mice after treatment
with CL316,243. Similar decreases in
MAT volume are observed in UCP1-Dta
mice, however, the presence of Dta does
not influence this effect. *p<0.05
Figure 6. The
mTmG reporter is
activated in the
inguinal WAT of
Adiponectin-Cre x
mTmG mice
(positive control)
(A) and is
selectively activated
in inguinal WAT of
UCP1-Cre x mTmG
mice after CL316
treatment (B).
BMAT in the tail
vertebrae is activated
in Adiponectin-Cre
mice (C), but there
appears to be no
Cre-mediated
activation in UCP1-
Cre x mTmG mice
(D). 20x
magnification. Scale
bar = 100 µm.
A
D
Figure 4. (A) Osmium-stained
tibiae were scanned by µCT and
reconstructed with the decalcified
bone overlaid. Quantification of
tibial MAT volume (N = 6 WT (4-
week-old), 7 UCP1-Dta (4-week-
old), 10 WT (16-week-old), and 7
UCP1-Dta (16-week-old) from the
first 100 – 20 µm slices distal to
the growth plate (B) and the total
tibial length (C) demonstrate a
trending decrease in age-related
accumulation of BMAT. This
decrease in MAT volume of the
UCP1-Dta mice may be due to an
effect from an exercise phenotype
that increases movement in the XY-
projection (D) while decreasing
rearing behavior in the Z-
projection (E).
A B
Figure 2. (A) 3D reconstruction from a focused ion beam scanning electron
microscopy (FIB-SEM) dataset showed a large lipid droplet with smaller
lipid droplets enmeshed by a dense mitochondrial network [1]. (B) Adult
mice were treated with CL316,243, a β3-adrenergic agonist for 72-hours to
induce beiging. In BMAT, remodeling was noted in a subset of adipocytes in
female, but not male mice [2]. Perilipin in red. DAPI in blue. Solid white
box shows a magnified inset of remodeling.
Activation of the mTmG reporter by UCP1-Cre in adipocytes of WAT indicate
these are beige adipocytes. However, lack of activation in the BMAT suggest
they are their own unique population of adipocytes.
WAT
BMAT
BAT
Adiponectin-Cre+ x mTmG UCP1-Cre+ x mTmG
iWAT
BMAT
A
B
C
D
A B
A
Dark grey = MAT; Light grey = demineralized bone
B
C
D
E
*h
*h