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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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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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, mstyner@med.unc.edu

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

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150

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600

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(%)

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1

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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

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(mm

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20

40

60

80

100

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Ar

(%)

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2

4

6

8

10

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m (

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*

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2

4

6

8

10

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m (

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**

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1

2

3

4

5

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m)

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0.02

0.04

0.06

Tb

.Th

(m

m)

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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

ACC

EPTE

D M

ANU

SCR

IPT

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