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Bone marrow fat accumulation accelerated by high fat diet is suppressed byexercise
Maya Styner, William R. Thompson, Kornelia Galior, Gunes Uzer, XinWu, Sanjay Kadari, Natasha Case, Zhihui Xie, Buer Sen, Andrew Romaine,Gabriel M. Pagnotti, Clinton T. Rubin, Martin A. Styner, Mark C. Horowitz,Janet Rubin
PII: S8756-3282(14)00118-5DOI: doi: 10.1016/j.bone.2014.03.044Reference: BON 10309
To appear in: Bone
Received date: 30 December 2013Revised date: 17 February 2014Accepted date: 25 March 2014
Please cite this article as: Styner Maya, Thompson William R., Galior Kornelia, UzerGunes, Wu Xin, Kadari Sanjay, Case Natasha, Xie Zhihui, Sen Buer, Romaine Andrew,Pagnotti Gabriel M., Rubin Clinton T., Styner Martin A., Horowitz Mark C., RubinJanet, Bone marrow fat accumulation accelerated by high fat diet is suppressed by exer-cise, Bone (2014), doi: 10.1016/j.bone.2014.03.044
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Bone marrow fat accumulation accelerated by high fat diet is suppressed by
exercise
Maya Styner1, William R. Thompson1, Kornelia Galior1, Gunes Uzer1, Xin Wu1, Sanjay
Kadari1, Natasha Case1, Zhihui Xie1, Buer Sen1, Andrew Romaine3, Gabriel M.
Pagnotti4, Clinton T. Rubin4, Martin A. Styner2, 3, Mark C. Horowitz5, Janet Rubin1
1Department of Medicine, University of North Carolina, Chapel Hill, NC
2Department of Computer Science, University of North Carolina, Chapel Hill, NC
3Department of Psychiatry and Computer Science, University of North Carolina, Chapel
Hill, NC
4Department of Biomedical Engineering, State University of New York, Stony Brook, NY
5Department of Orthopedics and Rehabilitation, Yale University, New Haven, CT
Corresponding author: Maya Styner, University of North Carolina department of
medicine, division of Endocrinology, CB 7170, 5002 Burnett Womack, 160 Dental
Circle, Chapel Hill, NC 27599-7170, +1-919-843-0711, [email protected]
Funding: MS: AR062097, JR: AR042360, AR056655, CR: AR 43598, EB 14351, MH:
DK092759
Abbreviations: MAT, marrow adipose tissue; RD, regular diet; HFD, high fat diet; RD-
E, regular diet exercise; HFD-E, high fat diet exercise; trabecular BV/TV, trabecular
bone volume/total volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp,
trabecular separation; Tt.Ar, total cross sectional area in the periosteal envelope; Ct.Ar,
cortical bone area; Ct.Ar/Tt.Ar, cortical area fraction; Ct.Th, average cortical thickness.
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Abstract
Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic
insufficiency, remains poorly understood and difficult to quantify. We tested the
response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis,
and compared it to measures of bone quantity. We hypothesized that HFD would
increase MAT and diminish bone quantity, while exercise would slow MAT acquisition
and promote bone formation. Eight week-old female C57BL/6 mice were fed a regular
(RD) or HFD, and exercise groups were provided voluntary access to running wheels
(RD-E, HFD-E). Femoral MAT was assessed by µCT (lipid binder osmium) using a
semi-automated approach employing rigid co-alignment, regional bone masks and was
normalized for total femoral volume (TV) of the bone compartment. MAT was 2.6-fold
higher in HFD relative to RD mice. Exercise suppressed MAT in RD-E mice by more
than half compared with RD. Running similarly inhibited MAT acquisition in HFD mice.
Exercise significantly increased bone quantity in both diet groups. Thus, HFD caused
significant accumulation of MAT; importantly running exercise limited MAT acquisition
while promoting bone formation during both diets. That MAT is exquisitely responsive to
diet and exercise, and its regulation by exercise appears to be inversely proportional to
effects on exercise induced bone formation, is relevant for an aging and sedentary
population.
Highlights
• High fat diet (HFD) increases marrow adipose tissue (MAT) similar to diet effect on
visceral white fat depots.
• Running exercise significantly prevents MAT accumulation in regular diet and HFD-
fed mice.
• Exercise limitation of MAT is associated with an increase in bone quantity.
• Reliable quantification and visualization of MAT in the mouse femur is demonstrated
via osmium stain and volumetric micro-CT image analysis.
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Keywords: Exercise, Marrow Adipose Tissue, Micro-CT, Quantitative Image Analysis,
Bone Micro Architecture, Lipid
1.1 Introduction
As obesity and its associated metabolic sequelae reach epidemic proportions
globally, considerable effort has been invested in understanding the distinct roles of
specific adipose depots in contributing to disease states. Besides the fat accumulating
in visceral and subcutaneous depots, adipose tissue found in the bone marrow space
(marrow adipose tissue; MAT) is of clinical interest. The adipocytes that form MAT are
generated in the bone marrow from mesenchymal stem cells (MSC), and are more
closely related to osteoblasts than other cells of mesenchymal origin [1]. Both mouse
and human studies indicate that conditions of increased bone formation are associated
with decreased marrow fat [2, 3], an inverse relationship thought to be due to the
preferential allocation of MSC into bone forming osteoblasts rather than adipocytes [4].
Human data supports a clinical relationship between increased MAT and low bone
mass; this has been demonstrated in anorexia nervosa [5], paraplegia [6] and post-
menopausal osteoporosis [7]. In a study of young adults, marrow fat was inversely
correlated with measures of vertebral bone density and femoral cortical bone area [8].
Further, as precursors for MAT are critical components of the hematopoietic-MSC
niche, an alteration in MAT is likely to influence MSC function, either through local
autocrine effects, or through physical encroachment [9], which could compromise the
ability to regenerate damaged connective tissues.
The physiology of MAT is poorly understood, both in terms of its dependence and
effect on MSC lineage allocation and as a potential storage depot for excess calories
[10]. Importantly, whether excess calories or calorie expenditure during exercise can
regulate the quantity of MAT, as it does in non-marrow adipose depots, is unknown. A
recent positive correlation between bone marrow fat and intrahepatic lipid,
intramyocellular lipid, and serum triglyceride level suggests that MAT might serve as a
storage depot for excess calories [11]. MAT has a unique composition of lipid species
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[1] and recently has been shown to have a gene expression profile that overlaps with
brown as well as white adipose tissue [12]. Interestingly, starvation diets appear to
increase MAT depots [13, 14] but how high fat diet affects this lipid compartment is
unclear.
Exercise is universally recognized as a means of suppressing obesity-associated
white adipose tissue depots as well as enhancing bone density and muscle mass [15-
18]. Skeletal loading stimulates bone formation as has been widely shown in humans
[19] and animals [20-22]. Our laboratory has demonstrated that mechanical input,
analogous to exercise, promotes cytoskeletal complexity and activation of βcatenin in-
vitro, signals associated with osteoblastogenesis [23-31]. These effects translate to the
in-vivo physiology as well, as MSC extracted from exercising mice have increased
βcatenin and a reduced capacity for adipogenesis [20].
The present study was designed to test the effects of diet and exercise on MAT and
bone. We hypothesized that a high fat diet would increase MAT analogous to increases
expected in white adipose tissue, and thus MAT might, besides representing a
pathologic diversion of MSC into adipocytes, function as an energy storage depot.
Further, we asked if changes in MAT would correlate with measures of bone quantity.
To answer whether diet and exercise affect MAT, we applied a quantitative, volumetric
method for measuring and localizing marrow adiposity [32]. This method identified the
presence of lipid by its ability to bind osmium, a method widely used to identify adipose
tissue [33, 34]. As osmium signal can be separated from both marrow and bone by a
high CT density [35], we were able to analyze µCT images to obtain volumetric
assessment of marrow fat.
Here we quantified MAT in mice fed regular or high-fat diets and provided access to
voluntary wheel running in exercise groups. We show that diet and exercise strongly
influence MAT quantity in opposite directions. While the exercise reduction in MAT was
associated with increased bone quality, the increased MAT due to six weeks of high fat
diet did not adversely affect bone. Our results are the first to quantify an increase in
MAT in response to diet, and a reduced accumulation of MAT in response to running
exercise, changes that were inversely correlated to bone quantity.
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1.2 Materials and Methods
Animals and Diet: The UNC IACUC approved the use and care of animals in the
study. Eight-week old female C57BL/6 mice (n = 20) were randomly assigned to one of
two diets for a period of 6 weeks: 1) regular diet, RD, low in fat (PicoLab Mouse Diet 20,
Item #: 5058) or 2) a high-fat diet, HFD diet comprised of 45% fat, 35% carbohydrate
and 20% protein, with the majority of fat calories derived from lard (Research diets, DIO
Series Diets Item #12541). Mice were fed ad libitum; the grams of food consumed were
weighed and calories per gram calculated. In a second study, 10-week old, skeletally
mature, C57BL/6 mice were studied to assess effects of exercise on marrow adiposity.
Female C57BL/6 mice were used because this gender and strain has been shown to be
highly motivated to run within the voluntary wheel running protocols [36].
Exercise Intervention: Mice in either diet group were allocated to an exercise
intervention for 6 weeks. The groups were as follows: 1) Regular Diet, RD (n = 5), 2)
Regular Diet Exercise, RD-E (n=5), 3) High fat, HFD (n=5), 4) High Fat Exercise, HFD-E
(n=5). Change to HFD and/or access to the exercise wheel were commenced
simultaneously. Both control and running mice were individually housed for the duration
of the running experiments so that running could be tracked for each mouse. Food
consumption and exercise was voluntary, mice were weighed weekly. A voluntary,
running wheel exercise intervention was used as previously described [36]. The wheels
were suspended above the cage floor by metal brackets. Exercise wheel use was
monitored using a Mity 8 Cyclocomputer (model CC-MT400), recording distance,
average speed, and daily running time. Control mice were housed similarly (one
mouse/cage) but did not have wheel access.
Specimen harvest and preparation: Running wheels were removed 24 hours prior to
harvest. Animals were weighed and euthanized by CO2 inhalation followed by
decapitation. Perigonadal fat pads were collected and weight normalized to body
weight. The visceral fat pad that is the female-equivalent for the epididymal fat pad is
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the perigonadal fat pad (also known as the parametrial fat pad[37]). Right and left femur
and tibia were harvested and soft tissue dissected away, tibiae were prepared for µCT
imaging by fixation with 10% neutral buffered formalin at 40C overnight as previously
described [38]. Femur specimens were prepared for MAT osmium stain by fixation with
10% neutral buffered formalin at 40C overnight, followed by decalcification with 14%
EDTA for 14 days as previously described [35]. The contralateral femur was snap
frozen for triglyceride assay.
Triglyceride Assay: Each mouse femur was crushed using mortar and pestle into the
Cayman TG assay lysis buffer along with the addition of the protease/phosphatase
inhibitors (Halt Protease Inhibitor Cocktail from Thermo scientific). After
homogenization and centrifugation per the manufacturer instructions, the supernatant
was analyzed for triglyceride content using Cayman's TG Assay Kit (Cayman Item
Number 10010303). The triglyceride content was determined for each mouse femur
and normalized to total protein content.
Quantification/imaging of MAT: Osmium staining of lipid in mouse bones has been
previously described[32, 35]. Briefly, femurs were fixed with 10% neutral buffered
formalin (Fisher #SF100-4) for 24 hours at 40C. The next day, specimens were rinsed
with water for 1hr. Specimens were then decalcified in 14% EDTA at 40C for 14 days.
Femurs were rinsed in water and cut just below the femoral head. The bones were
incubated with 1% osmium tetroxide/2.5% potassium dichromate for 48 hours at room
temperature. Specimens were placed in cassettes and washed in water for 2 hrs.
Imaging was performed via µCT with energy of 55kVp, integration time of 500
milliseconds, and resolution of 0.01 x 0.01 x 0.01mm (Scanco µCT-35, Scanco Medical,
Bruttisellen, Switzerland). Raw, unaligned femur images were first rigidly aligned (figure
1A and 1B, 3D Slicer, www.slicer.org) [39] to present all images in a consistent fashion
and to allow for superimposition of images. Bone masks were created next, starting
from segmentation via standardized bone thresholds, manual contouring was performed
to outline the femur in all images to designate a total femoral volume (Insight-SNAP,
www.itksnap.org) [40] (figure 1C). As osmium is significantly more dense than bone,
Hounsfield Unit (HU) thresholds were set for visualization of osmium as follows: low
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osmium from 2000-3000 HU (figure 1E/F red), mid osmium from 3000-4000 HU (figure
1E/F green) and high osmium from 4000-5000 HU (figure 1E/F blue). The lower
threshold for osmium was chosen to be considerably above the HU for dense cortical
bone [41, 42]. The contribution of potentially mislabeled cortical bone to the osmium
volume is expected to be negligible [35]. The maximal threshold of 5000 HU was
chosen by inspecting the 99th percentile over all image intensities. In between the lower
and upper threshold, we divided the osmium attenuation range into three uniformly
separated categories.
Osmium within the bone mask was then quantified as volumetric (mm3)
measurements of low, mid and high osmium stain regions in the femur. Finally, the
femur was subdivided into anatomical regions of interest: a) epiphysis: distal femur from
knee joint to growth plate primary center of ossification (figure 1G green), b)
metaphysis: distal shaft from growth plate to the distal third of the shaft as measured by
the shaft length in the average image (figure 1G red) and c) diaphysis: proximal
remainder of the shaft (figure 1G blue). Regional osmium volume normalized to total
femoral volume TV (as % of TV) was assessed. We reported and analyzed the %
osmium (osmium in mm3/total volume) above the mid osmium threshold of 3000 HU
(figure 1H). Of note, if the higher threshold of 4000 HU was applied, the effects of diet
and exercise were similar to those reported at 3000 HU. Aligned femoral images were
averaged across all images to form an average femur image as a reference for bone
visualization (figure 1D). Images were also averaged within each group separately for
color-coded visualizations of the osmium densities in order to allow for visual
comparisons of MAT between groups (figure 1E). The distribution and location of MAT
has been previously described in [9] and was validated in RD and HFD animals using
histology (supplemental figure 1).
Bone microarchitecture: Bone morphology parameters of the proximal tibial
metaphysis and mid diaphysis were quantified ex vivo using high-resolution X-ray
micro-computed tomography (µCT, Scanco Medical; Wayne, PA). These included bone
volume (BV), bone volume fraction (BV/TV), trabecular number, thickness and spacing,
and measures of cortical area and thickness. Beginning 300µm distal to the growth
plate, 1200µm of the metaphysis was evaluated at 12µm resolution and 55keV intensity
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settings. A threshold for each slice was set exclusively for cortical and trabecular bone
using an automated script [43]. The reconstructed solid 3D images were then used to
quantify bone microarchitecture.
Statistics: Results are expressed as mean ± SD unless otherwise specified. Statistical
significance was evaluated by 2-tailed unpaired t-test or ANOVA with post hoc test
(GraphPad Prism 5.04). Statistical significance is indicated on graphs and figure
legends as *P < 0.05, **P < 0.01, and ***P < 0.001.
1.3 Results
Running distance, time and speed was similar in mice on HFD and regular chow
diet
Mice began running within 48 hours of exposure to running wheels, with the majority
of running hours during the night cycle. Daily running times and distances were
consistent with previously published studies on voluntary wheel based running [36].
There was little variability between individual mice in use of the running wheel; mice ran
at least 5 hours each 24-hour period, and every single day (Table 1). Mice eating the
regular diet (RD) ran a daily average of 358 ± 65 minutes, a time that was not
significantly affected by feeding of the HFD diet, at 367 ± 54 minutes daily. This was an
average of 10.9 ± 3.2 kilometers and 11.0 ± 1.6 respectively for RD-E and HFD-E mice.
The average speed was 2.02 ± 0.38 (RD-E) and 2.14 ± .09 (HFD-E) km/hr. There was
no significant difference between RD-E and HFD-E mice with regards to running time,
distance or average speed.
Six weeks of running did not significantly alter white adipose tissue
We examined the effect of 6 weeks of ad libitum high fat feeding on young female
mice (HFD diet starting at 8 weeks). In our facility, RD and HFD fed mice consumed
similar daily calories, as measured by daily weight of food consumed (figure 2B), as the
animals limited grams consumed of the calorie-dense high fat chow (figure 2A). Six
weeks after the diet change, (figure 2C), HFD-fed mice weighed 20.5 ± 2.4 grams, a
9% increase over control mice (18.8 ± 1.2 grams, reported as mean ± SD); however this
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was not a significant difference. Weights of HFD-fed and chow-fed mice begin to
diverge after 6-8 weeks of high fat feeding, and can diverge earlier if HFD diet is
initiated upon weaning [44, 45]. The fact that weights of the HFD group in our study
were not statistically significant after 6 weeks might be due to gender (we used female
C57BL/6 as opposed to males used in [44, 45]) or the specific formulation of the control
diet (our study used PicoLab Mouse Diet 20. The Pico-lab control diet contained 21%
fat-calories in contrast to the low-fat diet which contained 10% fat-calories used in [44],
the 7% fat-calorie diet used in [45]. As such, our control animals may have been
heavier due to fat calories, preventing early assessment of increased total body weight.
HFD-E mice ate similar grams of chow as the non-runners (130.0 g ± 6.4 vs. 113 g ±
14.6 reported as mean ±SD) with a trend for increased calories in exercisers (figure 2B,
p=ns). At the end of the experiment the weights of HFD non-runners and HFD-E were
not statistically different (HFD 20.5 ± 1.1g v. HFD-E 21.4 ± 0.9g, p=ns). The only
statistically significant divergence in weight was between the RD runners and non-
runners, with runners weighing nearly 4 grams more (RD 18.8 ± 1.2 g v. RDE 22.3 ±
2.6, figure 2C).
Excised perigonadal fat pads were normalized to total body weight to control for
variations in individual mouse size. Both groups of HFD fed mice showed significant
increases in the perigonadal fat weight normalized to body weight (figure 2D, p<0.05).
Perigonadal fat percentage increased by 46% in the setting of HFD diet (figure 2D,
p<0.05). Running did not attenuate perigonadal fat percent in either RD-E or HFD-E at
the 6-week time period of the experiment (percent change of -0.4% for RDE vs RD and
+6.0% for HFD-E vs HFD, p=ns for both, figure 2D).
Exercise suppressed marrow adipose tissue (MAT) despite high fat diet
MAT was analyzed using volumetric µCT assessment of the osmium stain from the
proximal diaphysis to the distal epiphysis (figure 1). Qualitative assessment was
achieved by viewing average images of the femur (n=5 per group) superimposed with
color-coded osmium density maps, presented in the sagittal, coronal and axial plane.
The sagittal and coronal plane images demonstrated MAT at the level of the growth
plate and into the shaft of the diaphysis (figures 3A,B). Distal to the growth plate, a
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reduced amount of osmium was detected. However, relative to the distal epiphysis,
increased osmium staining was noted in the metaphysis. The osmium density images
allow localization of fat throughout the full bone suggesting significant spatial variation
when viewed across axial slices (figure 3C). To be clear, this is not a representative
image: each image in figure 3 is an averaged image of n=5 bones per group obtained
by overlaying images. As noted in methods, the data shown is osmium percent of total
femoral volume (TV) above a threshold of 3000 HU. In order to visualize the distribution
of MAT, we present the images in the sagittal (figure 3A), coronal (figure 3B) and axial
(figure 3D) planes. This data, analyzed by region, is shown in detail in Table 2.
At all sites imaged, HFD stimulated a significant increase in MAT compared to that in
mice fed a control diet. Selecting the metaphysis, volumetric MAT increased from 8.5 ±
3.4 % osmium volume/total volume to 22 ± 8.2 (Table 2 and figure 3E, p<0.05). Figure
3E shows that HFD caused and increase in MAT to 2.6-fold relative to RD. The increase
in the marrow compartment was similar to diet-induced increases in perigonadal fat
(figure 2D). As such, MAT was found to be highly responsive to dietary fat feeding.
In animals fed RD, exercise significantly reduced accumulation of MAT. The MAT in
the imaged femur (epiphysis + metaphysis + diaphysis) in runners was half that
measured in non-runners (Table 2, p<0.05). Running prevented MAT accumulation at
all sites except the diaphysis in RD-E. The change at the metaphysis is shown in figure
3E with mean ± SD as in Table 2 (p<0.05).
Exercise reduction in MAT was even more robust when MAT acquisition was
stimulated by HFD. High fat diet increased MAT by 2.6-fold in sedentary animals
(compare HFD to RD); exercise almost entirely prevented this dietary increase.
Comparing HFD-E to HFD sedentary, exercise reduced MAT in the metaphysis by 2.3-
fold (figure 3E, p<0.05).
MAT quantity in non-metaphyseal sites also responded to both diet and exercise.
The increase in MAT due to dietary fat was visualized in all regions of the distal femur
proximal to the growth plate with the exception of the diaphysis, which appeared to be
spared from increases in MAT due to diet, and unaffected by exercise (Table 2). The
diaphyseal region had very little MAT as measured by osmium in either diet group,
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which may limit any valuation of change. Epiphyseal MAT also increased with HFD
feeding, but the running effect on MAT in the epiphysis did not reach statistical
significance (1.5-fold less in RDE vs. RD, 1.4-fold less in HFD-E vs. HFD, table 2),
again perhaps limited by the small amounts of volumetric fat found in this compartment.
Biochemical assessment of triglyceride/protein in the distal femur was increased in
HFD-fed mice, correlating with MAT/osmium data, but did not reach statistical
significance (figure 3F, triglyceride level was 274 ± 72 in RD vs 226.4 ± 67 in RDE and
314 ± 68 in HFD vs 276 ± 52 in HFD-E). Exercise, in agreement with the volumetric
MAT measure, resulted in a trend toward reduced triglyceride/femur protein
MAT response to exercise in skeletally mature mice
We next wished to ascertain if mice that had reached adulthood would use the
running wheel as much as when allowed access earlier, and to respond to exercise in
terms of limitation of MAT. We introduced 10-week old C54BL/6 females fed a regular
RD diet to voluntary running wheels. Adult females began running within 48 h, running
similar times and distances to their younger counterparts (Table 1, 349 ± 64 min, 12.0 ±
2.3 km). There was little variability between the times individual animals spent on the
exercise wheel, stressing that mice will run if given the opportunity.
In this experiment MAT was again significantly affected by exercise. Exercising mice
had MAT volume/metaphyseal volume that was 2.1-fold less than that in non-exercising
animals (Table 2 and figure 5A, p<0.05). Shown in figure 5B, the MAT response to
exercise was as robust as that in younger animals.
Running in regular and high fat fed mice increases bone quantity
Bone quantity was measured in the proximal tibial metaphysis and diaphysis to
assess effects of 6 weeks of HFD and exercise. HFD-fed mice had similar trabecular
bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness
(Tb.Th) and trabecular separation (Tb.Sp) compared to mice fed RD, and equivalent
cortical measures (Figure 4, Table 3). Six weeks of daily running induced increases in
trabecular bone volume of RD mice (RD 10±0.97% vs. RD-E 14±1.9%, p<0.05, table 3).
Moreover, running increased trabecular bone volume in HFD mice (HFD 9.2 ± 1.5 vs.
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HFD-E 13 ± 0.74, p<0.05, table 3). Trabecular thickness was 12% higher (p<0.01) in
RD-E compared with RD controls. In this time frame there were no significant
differences in Tb.Sp or Tb.N due to exercise; however a trend upward correlates with
the changes noted in trabecular BV/TV and trabecular thickness. Cortical bone area
was 0.97±0.066 in RD and 1.1±0.080 in RD-E (p<0.05, table 3). In the HFD mice, there
was a trend for increased cortical bone area with running but this was not significant.
The cortical area fraction was similar between groups. Both the periosteal perimeter and
the endocortical perimeter were increased in the runners (table 3). These data showed
that 6 weeks of running had significant effects on bone quantity irrespective of the type
of diet.
1.4 Discussion
Growing consensus links bone marrow adiposity with bone fragility [1] as exemplified
in clinical settings including skeletal unloading [46, 47], aging [48], postmenopausal
osteoporosis [49], and anorexia nervosa [13]. Imprecise quantification has previously
limited assessment of changes in MAT in response to potential regulators. Here we
have used a sensitive volumetric measure of marrow adiposity to demonstrate that MAT
quantity is sensitive to regulation by both diet and exercise. MAT rises rapidly in
response to high fat feeding without short-term effects on bone quality, and exercise
induction of bone formation prevents formation of MAT. This suggests that MAT might
represent an energy storage depot that can be limited by vigorous exercise.
Mice fed a high fat diet showed significant increases in marrow adiposity compared
to regular diet controls that were at least as great as diet induced increases in
perigonadal white adipose tissue. Surprisingly the significant increase in MAT preceded
that of generalized obesity; after 6 weeks there was only a trend toward obesity in high-
fat fed animals. Speculating on the rapid rise in MAT as compared to that of generalized
obesity, it is possible that the increase in MAT might represent high fat calorie
stimulation of marrow MSC to become adipocytes. Alternatively, it could be that lipid
accumulates in pre-existing marrow fat cells. With the ability to carefully quantify
adipose in the bone marrow, these questions can start to be addressed.
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Until recently, qualitative measurements of MAT have relied on bone histology [50,
51], which cannot adequately measure the volume of fat in the marrow and is subject to
site selection bias. Proton magnetic resonance spectroscopy (1H-MRS) has shown that
vertebral bone marrow fat parallels visceral fat in obesity in humans [52] and has
associated marrow adiposity with decreased bone mass [14]. Although 1H-MRS has
been used with some success to quantify vertebral MAT in humans [52], it is more
difficult in laboratory animals [53]. Furthermore, our µCT-based method demonstrates
superior resolution and specificity of distinguishing lipid from non-lipid tissue with
osmium binding, and producing an enhanced localization effect. Assessment of marrow
density by CT can also be used to estimate marrow fat, and has been used to support
an effect of impact loading to improve bone quality while decreasing marrow adiposity
[54]. However, local marrow density is not associated with cortical bone size,
underscoring the problems with estimating MAT from localized cross-sections. The
osmium-based technique used in our study further allows a focus on continuous, whole
regions of bone quantified using a volumetric approach. Osmium binds avidly to lipid
[55], allowing lipid to be demarcated from other components of the bone marrow space.
This is advantageous when compared to other imaging modalities, which do not
adequately distinguish between fat and hematopoietic compartments [56]. Additionally
because the density of osmium is significantly greater than that of demineralized bone
tissue stained with potassium dichromate, a clear separation between fat and bone
compartments permits specific and accurate quantification of adipose volume. An
additional advantage of the µCT imaging is the high resolution (commonly ~10µm
isotropic resolution) as compared with MR based imaging (commonly ~75-100µm
isotropic resolution) [57, 58]. In our study we were able to visualize increases in MAT in
the metaphysis extending into the diaphysis during high fat feeding, correlating with
histology and other data in the literature, indicating that marrow adipose normally
shares space within the trabecular bone [9, 59]. Further, in contrast to MR
spectroscopy, µCT imaging is pre-calibrated [60] and does not necessitate sophisticated
calibration [61]. As such, the osmium-µCT-volumetric analysis of MAT should prove to
be an invaluable tool in understanding MAT in rodent models.
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Bone quantity, in terms of trabecular and cortical volume, was not impaired by 6
weeks of high fat diet in growing animals that had not yet achieved obesity in terms of
weight, despite the large increase in MAT. As the increase in trabecular bone due to
exercise was equivalent between groups, the high fat diet did not appear to interfere
with bone formation. These short-term experiments do not rule out that MAT could
have chronic effects on bone quality. In comparison, while MAT accumulation was
limited by exercise, perigonadal white adipose tissue, which also increases with HFD,
was less affected, if at all, by running exercise. This suggests that these two fat depots
may be differentially regulated by both diet and exercise.
When marrow-derived mesenchymal cells are exposed to adipogenic stimuli in vitro,
they rapidly move toward adipocyte lineage [24, 28]. Mechanical signals restrict
adipogenesis through Wnt-independent [24] and cytoskeleton dependent [27, 29, 30]
generation of βcatenin. As mouse experiments have shown that βcatenin is activated
during loading [20, 62], it is possible that the effect of exercise on MAT is due to marrow
MSC lineage differentiation towards osteoblasts rather than adipocytes. Alternatively,
the decreased MAT accumulation during exercise might be due to energy expenditure.
Our volumetric technique cannot differentiate between these possibilities.
Reduced bone quality has been noted in obese individuals despite preserved bone
density measurements [63, 64]. This has significant clinical implications for exercise-
based therapies and our aging population, where being sedentary is the new normal.
That daily exercise suppressed the accumulation of MAT in mice could be interpreted to
mean that not having access to a running wheel may itself be enough to increase MAT.
Importantly, allowing mice to exercise at will, perhaps their true normal state, all but
prevented increases in MAT due to high fat feeding. This suggests that exercise can
“protect” against a marrow adiposity brought on by either HFD diet, aging or forced
sedentary behavior. Changes in MAT resulting from diet or exercise in humans have
not been measured due to technical limitations, but it is likely that our evidence showing
that these measures modify murine MAT would prove to be true in humans as well.
Our study has several limitations. Our experiments have not determined if MAT can
continue to increase within the marrow compartment, paralleling the almost unlimited
increase in extra-marrow white adipose tissue. Further, our finding that concurrent
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exercise limits the MAT accumulation due to high fat feeding does not answer whether
chronic increases in MAT can be minimized by exercise treatment.
In conclusion, MAT is a unique fat depot that responds rapidly to diet with increases
in quantity, as do other fat depots. MAT is, further, exquisitely sensitive to exercise in
growing and adult mice, changing more in short periods than do measures of bone. As
such, exercise prescriptions in populations susceptible to fracture may best be
quantified by alterations in marrow fat in the future. The mechanism by which diet or
exercise alter MAT is not known, but the ability to reliably quantitate this compartment
should allow future studies of its role in metabolism and its affect on bone formation.
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1.5 Figure legends
Figure 1. Overview of method for visualization and quantification of marrow
adipose tissue (MAT). Osmium-stained femorae were visualized via µCT. Femorae (A)
were rigidly aligned (B). Bone masks (C) were averaged (D). Osmium within the bone
mask was quantified as volumetric (mm3) measurements of low (red), mid (green) and
high (blue) osmium containing regions in the femur and (E) overlaid on µCT images for
viewing. 3D rendering of osmium regions (F) with same coloring as 3E, colors slightly
offset due to transparent bone mask. In (G) we subdivided the femur into three
anatomical regions of interest. (H) This is a pictorial representation of a data
spreadsheet containing regional osmium measurements as osmium volume normalized
to total femoral volume (in %).
Figure 2. White adipose tissue and weight are increased by HFD diet. C57BL/6
Mice had access to running wheels starting at the same time as the change in diet, age
8 wk. (A) Food intake. (B) Kcal consumed. (C) Body weight (D) Perigonadal fat pad
weight normalized to body weight. Results expressed as means ± SEM.
Figure 3. Exercise suppresses marrow adiposity despite HFD diet. 8 wk old
C57BL/6 mice were fed a RD or HFD diet for 6 weeks and given access to voluntary
running wheels. Visualization of osmium stain by µCT is demonstrated in the sagittal (A)
coronal (B), and axial (D) planes. (C) Osmium heat map and pictorial representation of
the anatomic planes demonstrated in A, B, D. Each image represents overlaid average
of n=5 mice. (E) Quantification of osmium in metaphysis as a measure of MAT. (F)
Triglyceride content of the femur (n=5 per group). Results expressed as means ± SEM.
Figure 4. Running increases bone quantity. Bone quantity was measured in the
proximal tibial metaphysis and diaphysis to assess effects of 6 weeks of HFD and
exercise via µCT.
Figure 5. Exercise reduces MAT in skeletally mature mice. Eight week-old and 10
week-old C57 BL/6 mice were fed a RD and provided access to running wheels (A)
Visualization of osmium stain by µCT is demonstrated in the sagittal and coronal planes.
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(C) Quantification of osmium in the metaphysis as a measure of MAT. Results
expressed as means ± SEM.
Supplemental figure 1. Histology demonstrates location and distribution of
marrrow fat. C57BL/6 mice were fed a RD or HFD diet for 6 weeks. Histologic sections
of the femur were performed and stained with H&E. Representative sections from 2
different RD and HFD mice are shown.
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1.6 References
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Mid Level HU 3000-4000
Figure 1. Methods overview
A. raw unaligned µCT B. aligned µCT C. masked µCT
D. average µCT
E. discrete osmium labels
F. osmium labels in 3D
Low Level HU 2000-3000
High Level HU 4000-5000
G. regions in 3D
H. regional volumes
proximaldiaphsysis
metaphysis
epiphysis
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Figure 2. White adipose tissue is increased by HFD.
weeks
weig
ht
(g)
0 1 2 3 4 5 6
16
18
20
22
24
26 RDRD-EHFDHFD-E
*ns
ns
ns
foo
d in
take (
g)
0
50
100
150
200
250
*
RDRD-EHFDHFD-E
kcal
0
200
400
600
800 * **
peri
go
nad
al
fat
(%)
0
1
2
3
***
ns
ns
A B
DC
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0
5
10
15
20
25
% o
sm
ium
meta
ph
ysis
*
*
***
Figure 3. Exercise supresses marrow adiposity despite HFD.
RD RD-E HFD HFD-E
A BSagittal Coronal
D AxialRD-ERD
HFD HFD-E
C
trig
lyeri
de
(µg
/ m
g o
f p
rote
in)
0
100
200
300
400
Low osmium HU 2000-3000
High osmiumHU 4000-5000
E
RD RD-E HFD HFD-E
RD RD-E HFD HFD-E
F
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Figure 4. Running increases bone quantity.
RD RDE RD
0
5
10
15
20
trab
ecu
lar
BV
/TV
(%
)
* *
0.0
0.5
1.0
1.5
Ct.
Ar
(mm
2)
p=0.08*
0.0
0.5
1.0
1.5
Tt.
Ar
(mm
2)
0
20
40
60
80
100
Ct.
Ar/
Tt.
Ar
(%)
0
2
4
6
8
10
Ps.P
m (
mm
)
*
0
2
4
6
8
10
Ec.P
m (
mm
)
**
0
1
2
3
4
5
Tb
.N (
1/m
m)
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0.02
0.04
0.06
Tb
.Th
(m
m)
**
0.0
0.1
0.2
0.3
Tb
.sp
(m
m)
RD
RD-E
HFD
HFD-E
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Figure 5. Exercise reduces MAT in skeletally mature mice.
HFDRun
ChowCTL
8 wk 10 wk sagittal
coronal
RD RD-E RD RD-E
RD RD-E RD RD-E
low osmium
high osmium
% o
sm
ium
m
eta
ph
ys
is
age 8wk age 10wk0
5
10
15
20RDRD-E
B
A
8 wk 10 wk
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Table 1. Running parameters (expressed as mean ± SD).
!
8 week 10 week
RD HFD RD P value
Average distance (km/day) 10.9 ± 3.2 11.0 ± 1.6 12.0 ± 2.3 P=0.75
Average time (min/day) 358 ± 65 367 ± 55 349 ± 64 P=0.90
Average speed (km/hr) 2.02 ± 0.38 2.14 ± 0.09 2.0 ± 0.21 P=0.731
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Table 2. Marrow adipose tissue (% osmium in mm3/total bone volume as mean ± SD).
8 week 10 week
RD RDE HFD HFD-E RD RDE
Epiphysis 1.6±0.53 1.1±0.32 7.4±5.5 5.3±2.2 0.85±0.34 1.5±1.9
Metaphysis 8.5±3.4 3.6±1.3
a 22±8.2
a 9.6±2.7
b 9.4±4.3 4.4±2.4
c
Diaphysis 1.4±1.4 1.3±1.1 0.94±0.30 0.62±0.26 0.14±0.089 0.04±0.055
Metaphysis+
diaphysis 6.0±2.0 2.9±1.3a 15±5.2
a 6.6±2.1
b 6.1±2.8 2.8±1.6
c
Total femur 5.0±1.6 2.4±1.1
a 13±5.1
a 6.2±1.8
b 5.0±2.0 2.6±0.89
c
a, p<0.05 compared with RD
b, p<0.05 compared with HFD
c, p<0.05 compared with Adult RD
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Table 3. Bone microarchitecture measurements by µCT (mean ± SD).
RD RD-E HFD HFD-E
Trabecular BV/TV
(%) 10±0.97 14±1.9a 9.2±1.5 13±0.74
b
Tb.N (1/mm) 4.2±0.35 4.6±0.13 4.0±0.22 4.2±0.20
Tb.Th (mm) 0.04±0.002 0.05±0.002a 0.04±0.004 0.05±0.005
Tb.Sp (mm) 0.24±0.02 0.22±0.009 0.26±0.022 0.24±0.013
Ct.Ar (mm2) 0.97±0.066 1.1±0.080a 0.97±0.066 1.1±0.089
Tt.Ar (mm2) 1.1±0.11 1.3±0.08 1.1±0.11 1.3±0.15
Ct.Ar/Tt.Ar (%) 85±3.1 86±2.0 85±3.2 83±3.0
Ct.Th (mm) 0.10±0.008 0.10±0.007 0.10±0.006 0.11±0.008
Ps.Pm (mm) 8.0±0.04 8.7±0.33a 8.2±0.15 8.5±0.20
Ec.Pm (mm) 7.6±0.46 8.4±0.32a 8.2±0.21 9.1±0.54
b
a, p<0.05 compared with RD
b, p<0.05 compared with HFD
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