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www.eje-online.org © 2017 European Society of EndocrinologyPrinted in Great Britain

Published by Bioscientifica Ltd.DOI: 10.1530/EJE-16-0448www.eje-online.org © 2017 European Society of Endocrinology

176:1 21–30I M de Araújo and others Marrow adipose tissue in type 2 diabetes

European Journal of Endocrinology (2017) 176, 21–30

176:1

10.1530/EJE-16-0448

Marrow adipose tissue spectrum in obesity and type 2 diabetes mellitusIana M de Araújo¹, Carlos E G Salmon², Andressa K Nahas³, Marcello H Nogueira-Barbosa¹, Jorge Elias Jr¹ and Francisco J A de Paula¹

1Department of Internal Medicine, Ribeirao Preto Medical School, 2Department of Physics, Faculty of Philosophy, Sciences and Arts of Ribeirao Preto, and 3Department of Epidemiology, School of Public Health, USP, Ribeirão Preto, Sao Paulo,, Brazil

Abstract

Objective: To assess the association of bone mass and marrow adipose tissue (MAT) with other fat depots, insulin

resistance, bone remodeling markers, adipokines and glucose control in type 2 diabetes and obesity.

Design and methods: The study groups comprised 24 controls (C), 26 obese (O) and 28 type 2 diabetes.

Dual-energy X-ray absorptiometry was used to determine bone mineral density (BMD). Blood samples were collected

for biochemical measurements. 1H Magnetic resonance spectroscopy was used to assess MAT in the L3 vertebra, and

abdominal magnetic resonance imaging was used to assess intrahepatic lipids in visceral (VAT) and subcutaneous

adipose tissue. Regression analysis models were used to test the association between parameters.

Results: At all sites tested, BMD was higher in type 2 diabetes than in O and C subjects. The C group showed lower

VAT values than the type 2 diabetes group and lower IHL than the O and type 2 diabetes groups. However, MAT was

similar in the 3 groups. Osteocalcin and C-terminal telopeptide of type 1 collagen were lower in type 2 diabetes than

those in C and O subjects. Moreover, at all sites, BMD was negatively associated with osteocalcin. No association was

observed between MAT and VAT. No relationship was observed among MAT and HOMA-IR, leptin, adiponectin or

Pref-1, but MAT was positively associated with glycated hemoglobin.

Conclusions: MAT is not a niche for fat accumulation under conditions of energy surplus and type 2 diabetes, also is

not associated with VAT or insulin resistance. MAT is associated with glycated hemoglobin.

Introduction

Obesity and type 2 diabetes mellitus are part of a spectrum respectively linked as cause and consequence, embedded in a metabolic environment of insulin resistance generated by adipose tissue and muscle. The performance of pancreatic β cells in insulin secretion determines the transition between normal glucose tolerance, pre-diabetes and type 2 diabetes, a typical pathway followed by obese individuals.

Obesity and type 2 diabetes are also independent risk factors for several disorders, including arterial hypertension, dyslipidemia, macroangiopathy and nonalcoholic steatohepatitis. These disorders have mechanisms closely related to insulin resistance, such

as visceral obesity, ectopic lipid deposition and an altered profile in circulating levels of adipokines (1). On the other hand, obesity and type 2 diabetes are usually associated with normal bone mass, albeit this feature does not necessarily mean that both have a protective effect against fractures (2, 3, 4). Several studies have shown that fracture risk is higher in obese postmenopausal women (5) and obese men (6) and that fracture risk is significantly higher in type 2 diabetes (7, 8). However, there are also data showing that, among obese individuals, fracture incidence is increased in those with low bone mineral density (BMD) (9).

Clinical Study

Correspondence should be addressed to F J A de Paula Email fjpaula@fmrp.usp.br

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The pathophysiology of bone disorder in obesity and especially type 2 diabetes most likely includes specific factors that do not pertain to the universe of other chronic complications usually associated with these metabolic diseases. Osteoblasts and adipocytes within the bone marrow originate from the same mesenchymal stem cell. Several lines of evidence indicate that nutritional deprivation influences the commitment of progenitor cells toward differentiation into osteoblasts or adipocytes, whereas far less is known about the effect of energy surplus.

The mechanisms related to higher fracture risk in obesity and type 2 diabetes have not been clearly delineated. A previous study reported derangement of collagen crosslinking and cortical porosity as possible mechanisms of decreased bone strength in type 2 diabetes (10). However, there is no consensus in the literature about the impact of insulin resistance, adipose tissue distribution and fat overflow to marrow adipose tissue (MAT) on BMD. Russel et al. reported that BMD has an inverse relationship with the rate of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in adolescent girls (11). There are also results suggesting that bone volume is negatively associated with MAT and VAT in obese individuals. On the other hand, improvement in insulin sensitivity does not necessarily lead to a beneficial effect on bone mass. For instance, both calorie restriction and treatment with thiazolidinediones have a positive effect on insulin action and glucose tolerance, but concomitantly provoke bone loss and MAT enhancement. The relationship between serum insulin levels and insulin resistance and different niches of fat depot, including MAT, has been recently evaluated in non-diabetic women (12). The study showed an inverse relationship between VAT and intrahepatic lipid (IHL) and serum levels of insulin, but no relationship was observed between MAT and circulating insulin levels. Despite these lines of evidence, the effect of insulin resistance and type 2 diabetes on bone quantity and quality has not been assessed.

The focus of this study was to assess BMD and diverse fat depots in obese and type 2 diabetes individuals and to investigate the association of bone mass and MAT with VAT, IHL and insulin resistance. The second objective was to analyze the association of MAT with factors originating in adipocyte cell lines and osteoblasts (leptin, adiponectin, Pref-1 and osteocalcin) as well as with metabolic control.

Subjects and methods

Subjects

Our study group comprised 24 controls (C: 14 females and 10 males), 26 obese subjects (O: 16 females and 10 males) and 28 patients with type 2 diabetes (15 females and 13 males). The clinical characteristics of these groups are shown in Table  1. The study was approved by the Institutional Review Board of the Ribeirao Preto Medical School, USP (#1149/2012), and all subjects gave written informed consent to participate.

Exclusion criteria were pregnancy, presence of a chronic disease known to affect bone metabolism, abnormal thyroid functioning, hypothalamic or pituitary disorders, use of estrogen/oral contraceptive, hormone replacement therapy, glucocorticoid or osteoporosis therapy (bisphosphonates, denosumab, teriparatide, strontium ranelate and calcitonin).

Due to claustrophobia, 1 control, 1 obese and 5 type 2 diabetes subjects did not undergo any MRI examination. In addition, 3 individuals in the obese group and 4 in the type 2 diabetes group refused to complete the abdominal examination. Serum insulin levels were measured in 17 C and 17 O subjects, but not in type 2 diabetes individuals due to antidiabetic therapy.

Methods

Biochemical assessment

Blood samples were collected between 0800 and 0900 h after a 12-h overnight fast. Biochemical measurements (calcium, glucose, glycated hemoglobin, phosphorous, albumin, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine) were performed on the day of blood collection. Calcium, phosphorus, alkaline phosphatase, fasting glucose, albumin, AST, ALT and creatinine were determined using an automatic biochemistry analyzer (Wiener lab, CT 600 i, Thermo Fisher Scientific). The levels of glycated hemoglobin (HbA1c) were measured by high-performance liquid chromatography (D10 – Hemoglobin A1C Testing System, Bio Rad). The serum aliquots for the other parameters were stored at −70°C until the day of the assay.

25-Hydroxyvitamin D (25 (OH) D) (Liaison, DiaSorin, Saluggia VC, Italy), intact PTH (Immulite I, Siemens) and IGF-I (Immulite 2000 Siemens) were determined

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y176:1 23Clinical Study I M de Araújo and others Marrow adipose tissue in

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by chemiluminescence. The serum levels of osteocalcin (host-Easia Diasource, Louvain-la-Neuve, Belgium), Pref-1 (Quantikine Human Pref-1 R&D Systems), leptin (Quidel, TECOmedical Group, Gewerbestrasse, Sissach, Switzerland) and adiponectin (Millipore) were determined by enzyme immunoassay. The C-terminal telopeptide of type I collagen (CTX) was measured by electrochemiluminescence (Cobas E 411, Roche Diagnostics). All intra- and inter-assay coefficients of variation were lower than 10% and 20% respectively.

1H-MR spectroscopy of bone marrow in L3

The volunteers underwent spine MRI in a 1.5 T system (Philips ACHIEVA, Philips Medical Systems), as described previously (12).

Briefly, the subjects were positioned head first in the magnet bore in the prone position. A phased-array coil was positioned over the lumbar region. Sagittal T2-weighted fast spin echo acquisition was used as a reference for the spectroscopy voxel placement. A single

voxel of 3.37 mL was positioned in the center of the third lumbar (L3) vertebral body. The point resolved spectroscopy (PRESS) technique was applied using the following parameters: repetition time (TR) = 2000 ms, three echo times (TE) = 40/60/80 ms, 8 averages, without fat or water suppression.

MRS data were processed with LCModel software (Version 6.1, http://www.s-provencher.com/pages/lcmodel.shtml). The area values of the CH2 lipid peak at 1.3 ppm and the water peak at 4.7 ppm were T2-corrected using a fitting to a mono-exponential decay curve. Finally, the MAT content of L3 was expressed as lipid/(water + lipid) estimated from the corrected water and fat concentrations.

Abdominal magnetic resonance imaging

Abdominal images were acquired with a phased-array torso coil. A coronal turbo-spin-echo (TSE) T2-weighted sequence with breath-holding was applied to localize the following scan volumes. Consecutively, a breath-holding

Table 1 Clinical characteristics of the patients, biochemical measurements, BMD, MAT, VAT and IHL results.

Control (n = 14F/10M) Obese (n = 16F/10M) Type 2 diabetes (n = 15F/13M)

Age (years) 55 ± 7 52 ± 11 54 ± 8Body mass index (kg/m²) 23.9 ± 1.7* 30.8 ± 4.0 33.0 ± 7.3Height (m) 1.67 ± 0.1 1.64 ± 0.1 1.62 ± 0.1Weight (kg) 67 ± 8* 83 ± 17 86 ± 16Time of diagnostic (years) – – 11.8 ± 6.6Glucose level (mmol/L) 5.0 ± 0.3 5.0 ± 0.4 9.1 ± 4.5†

HbA1c (%) 5.6 ± 0.4 5.6 ± 0.3 9.2 ± 2.8†

HbA1c (mmol/mol) 37 ± 4 38 ± 4 77 ± 31†

HOMA-IR 1.73 ± 0.79 2.93 ± 2.18 –Calcium (mmol/L) 2.5 ± 0.1 2.5 ± 0.2 2.4 ± 0.1Phosphorus (mmol/L) 1.1 ± 0.2 1.1 ± 0.2 1.2 ± 0.3PTH (pmol/L) 3.78 ± 1.86 4.28 ± 1.86 3.78 ± 2.33Alkaline phosphatase (U/L) 165 ± 48 188 ± 55 180 ± 46IGF-1 (µg/L) 143 ± 46 130 ± 60 130 ± 4025(OH)D (mmol/L) 63 ± 23 56 ± 17 53 ± 20Osteocalcin (µg/L) 9.6 ± 3.8 9.4 ± 7.0 6.6 ± 2.8§

CTX (ng/L) 360 ± 160 390 ± 160 260 ± 90‡

Adiponectin (ng/mL) 15.1 ± 13.6 8.3 ± 4.1 7.4 ± 4.3▫

Leptin (µg/L) 25.3 ± 22.2 44.3 ± 25.4 31.7 ± 27.2Pref-1 (ng/mL) 0.27 ± 0.15 0.28 ± 0.12 0.32 ± 0.18L1–L4 BMD (g/cm²) 0.949 ± 0.109 0.972 ± 0.151 1.069 ± 0.141†

Total hip BMD (g/cm²) 0.911 ± 0.117 0.946 ± 0.129 1.046 ± 0.155†

Femoral neck BMD (g/cm²) 0.764 ± 0.113 0.816 ± 0.143 0.903 ± 0.174†

VAT (mm²) 7913 ± 4851¶ 10 774 ± 4614 11 349 ± 4899SAT (mm²) 20 753 ± 6721* 31 344 ± 12 594 28 544 ± 12 438IHL (%) 2.9 ± 3.6* 7.5 ± 7.2 11.2 ± 8.8MAT (%) 35.9 ± 9.3 31.8 ± 6.8 36.5 ± 8.4

BMD, bone mineral density; CTX, carboxy-terminal telopeptide of type I collagen; HbA1c, glycated hemoglobin; IGF-1, insulin-like growth factor 1; IHL, intrahepatic lipids; L1–L4, lumbar spine 1–4; MAT, marrow adipose tissue; Pref-1, Preadipocyte factor 1; PTH, parathyroid hormone; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue; 25(OH)D, 25-hydroxivitamin D.*C < type 2 diabetes and O; †type 2 diabetes > O and C; ‡type 2 diabetes < O and C; §type 2 diabetes < C; ▫C > type 2 diabetes and O; ¶type 2 diabetes > C.

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axial gradient double-echo T1-weighted sequence, in phase (echo time = 4.2 ms) and out of phase (echo time = 2.1 ms, slice thickness = 6.0 mm), was acquired including the whole abdomen, centered on the umbilical region.

The formula: fat = (SI in phase − SI out of phase)/(2SI − in phase) was used to calculate IHL, VAT and SAT from the averaged signal intensity (SI) in each region of interest (ROI) or labels defined by image segmentation. The SI values in the previously mentioned formula refer to the pair in/out phase images. In the liver, a manual segmentation was performed to select four ROIs as representative segments of the liver at the level of the main portal vein. The label of the visceral and subcutaneous fat areas was defined using the Display software (http://www.bic.mni.mcgill.ca/software/Display/Display.html) and a semiautomatic segmentation of an axial slice at the level of the umbilicus. This methodology has been described previously in detail elsewhere (12).

Dual-energy X-ray absorptiometry

Bone mineral density in the lumbar spine (L1–L4), total hip and femoral neck was determined by dual-energy X-ray absorptiometry (Hologic Discovery Wi, QDR series, Waltham, MA, USA). The precision error was 1.2% for L1–L4, 2.3% for the femoral neck and 2.7% for total hip. BMD values are expressed as g/cm2.

Statistical analysis

The data for the three groups were analyzed by one-way ANOVA followed by the Duncan post-test. The confidence interval was 95%. ANOVA was calculated with the aid of the SAS 9.4 software (SAS Institute Inc.,

SAS/STAT User’s Guide, Version 9.4, Cary, NC, USA: SAS Institute INC., 2013).

The linear regression model was applied, with age and BMI considered as covariates. To determine the association of the variables of interest, simple linear regression models (model 1) were adjusted by age and BMI (model 2), yielding the regression coefficients and R-square. All analyses were carried out using the SAS 9.4 software. The level of significance was set at 0.05.

Results

The study included 24 healthy controls, 26 obese subjects and 28 type 2 diabetes patients. Table  1 shows the anthropometric characteristics and biochemical evaluation of the subjects enrolled in this study. The O and type 2 diabetes groups were appropriately matched for age, sex, height, weight and BMI, but both showed higher weight and BMI than the C group. The circulating levels of glucose and glycated hemoglobin (HbA1c) were higher in the type 2 diabetes group. The three groups had similar serum levels of calcium, phosphorus and alkaline phosphatase.

No significant differences in the serum levels of ALT, AST, creatinine, PTH, 25(OH)D and IGF-I were observed between groups (Supplements). The C and O groups had similar serum levels of osteocalcin and CTX and their values were higher than those observed in type 2 diabetes patients.

BMD values for the lumbar spine (Fig. 1A), total hip (Fig. 1B) and femoral neck (Fig. 1C) were higher in type 2 diabetes than those in O and C subjects (P < 0.05), but did not differ between the O and C groups (Table 1). The number of individuals with osteopenia (C = 11, O = 12, type 2 diabetes = 9) and osteoporosis (C = 3, O = 3, type 2

Figure 1

Box-plots of (A) lumbar spine, (B) total

hip, (C) femoral neck bone mineral density

(BMD), (D) marrow adipose tissue (MAT),

(E) visceral adipose tissue (VAT) and (F)

intrahepatic lipids (IHL). *C < type 2

diabetes and O; †type 2 diabetes > O and

C; ¶type 2 diabetes > C.

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diabetes = 0) indicated that the rate of low bone mass was 58%, 57.6% and 32% in the C, O and type 2 diabetes groups respectively.

VAT was higher in type 2 diabetes than that in C subjects (Fig. 1E). Also, the type 2 diabetes and O groups showed significantly higher IHL than the C group (P < 0.05; Fig. 1F). In contrast, no difference in MAT values was found between the 3 groups (Table 1; Fig. 1F).

Table  2 shows the results of linear regression analysis. There was no association among lumbar spine BMD and IHL, VAT or VAT/SAT rate. Also, no

relationship was observed between BMD and serum insulin levels or HOMA-IR. A tendency to a negative association between BMD and MAT was observed in the lumbar spine (P = 0.061), but this weak relationship was not maintained after adjustment for age and BMI.

As expected, VAT and IHL exhibited a positive association (Fig. 2A), which persisted after adjustment for age and BMI. However, no association was observed between MAT and VAT (Fig. 2B) and VAT/SAT. Moreover, in contrast to VAT, MAT exhibited no relationship with HOMA-IR (Fig. 2C) or serum insulin levels.

Table 2 Linear regression results.

Associations

Model 1 Model 2

Estimate P value IC (95%) R2 Estimate P value IC (95%) R2

L1–L4 BMD × IHL 0.001 0.55 −0.003 0.006 0.01 −0.003 0.32 −0.003 −0.003 0.16Total hip BMD × IHL 0.003 0.19 −0.002 0.008 0.02 −0.001 0.73 −0.001 −0.001 0.18Femoral neck BMD × IHL 0.004 0.15 −0.001 0.008 0.03 −0.001 0.55 −0.001 −0.001 0.26L1–L4 BMD × VAT 2E−06 0.57 −1E−05 1E−05 0.01 −1E−06 0.7 −0.004 0.004 0.14Total hip BMD × VAT 1E−05 0.07 −1E−06 1E−05 0.05 3E−06 0.43 −0.146 0.146 0.18Femoral neck BMD × VAT 5E−06 0.21 −3E−06 1E−05 0.02 1E−06 0.88 −0.007 0.007 0.26L1–L4 BMD × VAT/SAT −0.016 0.81 −0.154 0.121 0.01 0.023 0.73 0.018 0.028 0.14Total hip BMD × VAT/SAT 0.067 0.33 −0.068 0.203 0.01 0.114 0.07 0.109 0.119 0.21Femoral neck BMD × VAT/SAT −0.013 0.85 −0.15 0.125 0.01 0.039 0.53 0.034 0.044 0.26L1–L4 BMD × insulin 0.0004 0.34 −5E−04 0.001 0.02 0.0001 0.91 −0.027 0.027 0.2Total hip BMD × insulin 0.0004 0.36 −4E−04 0.001 0.02 0.0002 0.63 −0.125 0.125 0.1Femoral neck BMD × insulin 0.0004 0.34 −5E−04 0.001 0.02 0.0001 0.91 −0.027 0.027 0.2L1–L4 BMD × HOMA-IR 0.022 0.08 −0.002 0.047 0.08 0.017 0.27 0.016 0.018 0.1Total hip BMD × HOMA-IR 0.019 0.08 −0.002 0.04 0.09 0.013 0.3 0.012 0.014 0.16Femoral neck BMD × HOMA-IR 0.018 0.14 −0.006 0.043 0.06 0.006 0.65 −0.116 0.128 0.22L1–L4 BMD × MAT −0.004 0.06 −0.008 0 0.05 −0.002 0.26 −0.003 −0.002 0.13Adiponectin × MAT −0.03 0.87 −0.34 0.29 0.01 −0.07 0.71 −0.42 0.29 0.05Adiponectin × VAT −7E−04 0.01 −0.001 −2E−04 0.13 −6E−04 0.02 −0.001 −1E−04 0.15Adiponectin × SAT 2E−05 0.83 −2E−04 0.0002 0.01 0.0006 <0.01 0.0002 0.001 0.21Adiponectin × VAT/SAT −13.06 <0.01 −22.48 −3.64 0.14 −15.25 <0.01 −24.56 −5.95 0.23Leptin × MAT −0.14 0.72 −0.93 0.65 0.01 0.17 0.68 −0.64 0.98 0.25Leptin × VAT −7E−04 0.39 −0.002 0.001 0.01 −0.002 0.02 −0.003 −2E−04 0.32Leptin × SAT 0.0015 <0.01 0.001 0.002 0.44 0.002 <0.01 0.001 0.0025 0.46Leptin × VAT/SAT −48.08 <0.01 −71.67 −24.49 0.24 −40.35 <0.01 −62.27 −18.43 0.4Pref-1 × MAT −0.003 0.18 −0.008 0.001 0.03 −0.004 0.12 −0.009 0.001 0.1Pref-1 × IHL 0.001 0.81 −0.01 0.01 0.01 −0.001 0.79 −0.01 0.01 0.03Pref-1 × VAT 1E−05 0.11 −1E−06 1E−05 0.04 5E−06 0.21 −3E−06 1E−05 0.06Pref-1 × VAT/SAT 0.03 0.64 −0.11 0.17 0.01 0.05 0.49 −0.09 0.19 0.04IHL × VAT 0.001 <0.01 0.0003 0.001 0.17 0.0005 0.011 −0.274 0.275 0.3MAT × VAT 1E−05 0.955 −4E−04 0.0004 0.01 0.0002 0.395 −7.275 7.275 0.18MAT × VAT/SAT 2.737 0.49 −4.999 10.473 0.01 0.667 0.85 −6.608 7.942 0.18MAT × HOMA-IR −0.055 0.16 −0.131 0.021 0.05 −0.008 0.83 −0.084 0.067 0.27MAT × insulin −0.857 0.34 −2.616 0.903 0.02 0.351 0.68 −1.342 2.043 0.31L1–L4 BMD × osteocalcin −0.012 <0.01 −0.019 −0.006 0.17 −0.011 <0.01 −0.015 −0.006 0.22Total hip BMD × osteocalcin −0.012 <0.01 −0.018 −0.005 0.15 −0.01 <0.01 −0.014 −0.005 0.21Femoral neck BMD × osteocalcin −0.013 <0.01 −0.02 −0.006 0.16 −0.011 <0.01 −0.015 −0.006 0.26Insulin × osteocalcin 0.751 0.58 −1.913 3.414 0.01 0.889 0.45 −1.397 3.175 0.31Osteocalcin × glucose −0.28 0.09 −0.6 0.05 0.04 −0.22 0.2 −0.55 0.11 0.08MAT × glucose 0.033 0.5 −0.063 0.129 0.01 0.057 0.28 −0.046 0.159 0.08MAT × HbA1c 0.321 0.4 −0.436 1.078 0.01 0.809 0.02 0.117 1.501 0.32

BMD, bone mineral density; HbA1c, glycated hemoglobin; IHL, intrahepatic lipids; L1–L4, lumbar spine; MAT, marrow adipose tissue; Pref-1, preadipocyte factor 1; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. Model 1 is a non-adjusted model and model 2 is a model with age and BMI considered as covariates.

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There were significant differences in the behavior of circulating levels of adipocyte-originated factors among the 3 groups. Although adiponectin was higher in C than that in O and type 2 diabetes subjects, the serum levels of leptin tended to be lower in the C group (P = 0.064). The 3 groups showed similar values of Pref-1. These 3 peptides produced by adipocyte cell lines showed no association with MAT (Table 2).

A negative association was detected among osteocalcin and lumbar spine (Fig. 2D), total hip (Fig. 2E) and femoral neck (Fig. 2F) BMD. However, no association was observed between the serum levels of osteocalcin and insulin or glucose.

MAT showed no association with glucose, but was positively associated with HbA1c.

Discussion

Obesity reflects the high capacity of white adipose tissue (WAT) to store lipids. However, the overflow of lipids from WAT to other tissues reveals that a certain limit exists concerning the ability of WAT to retain lipids in its domain. Excessive accumulation of fat within and subsequently outside adipose tissue leads to functional (insulin resistance) and structural disorders not only in adipose tissue itself but also in muscle, liver and pancreas (13). Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are illustrative consequences of lipotoxicity triggered by the overflow of lipids (14, 15). Bone disease in type 2 diabetes remains as a conundrum in which, unexpectedly, the role of MAT and insulin resistance has scarcely been investigated. This study suggests that MAT represents a diverse and sole type of adipose tissue: (a) it is not a site for fat storage during energy surplus, (b) it seems to have no relationship with VAT, serum levels of insulin

or insulin resistance and (c) is positively associated with HbA1c.

Several studies have observed that bone mass seems not to be negatively affected by type 2 diabetes and hyperglycemia (16, 17, 18). This study reaffirms these data and provides some additional interesting information. Bone mass was higher in type 2 diabetes not only compared with the control group but also compared with the obese group. These results indicate that BMD in type 2 diabetes individuals results from a more complex configuration than simply the mechanical and endocrine/paracrine influence of the adipose tissue existing in obese non-diabetic individuals. For instance, non-severely obese individuals do not share a low bone turnover profile with type 2 diabetes patients (19, 20, 21). Rochefort et al. (22) evaluated the serum concentrations of osteocalcin and insulin in a population of prepubertal obese and control children and observed that the circulating levels of osteocalcin were similar in the two groups. Similarly, another study conducted on non-diabetic overweight and obese adults reported that BMI and body weight did not differ across tertiles of total or undercarboxylated osteocalcin (23). On the other hand, the present results seem to conflict with those obtained in a previous study performed by Pittas et al. in a cohort study of individuals aged 65 years and older, showing that osteocalcin is inversely associated with BMI and fat mass (24). There are several differences between this study and the one by Pittas et al. which render them incompatible for comparison: the age difference between the two groups studied and, more importantly, the inclusion of some obese subjects who also had diabetes in the Pittas study. The present results agree with those reported in most studies of bone remodeling in type 2 diabetes, which identified low bone formation and resorption

Figure 2

Regression analysis among (A) lumbar

spine, (B) total hip and (C) femoral neck

bone mineral density (BMD) and

ostecalcin, (D) HOMA-IR and marrow

adipose tissue (MAT), (E) visceral adipose

tissue (VAT) and MAT, (F) intrahepatic

lipids (IHL) and MAT.

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activity based on the determination of biochemical markers (25, 26). Obese patients exhibited osteocalcin and CTX values similar to control. Moreover, there was a negative association between serum levels of osteocalcin and lumbar spine BMD, suggesting that the high BMD in type 2 diabetes is closely related to low bone turnover.

Although obesity is a major determinant of type 2 diabetes mellitus, the site of fat allocation is more important than the amount of fat (27). Several steps are involved in the onset of insulin resistance, especially the accumulation of fat in VAT and subsequently its redistribution to pancreas, muscle and liver (28). Next, β cell dysfunction becomes the last step in the onset of type 2 diabetes (29). Our data confirm previous studies showing that VAT and IHL are significantly increased in obese individuals (30) and even more so in type 2 diabetes patients (15). This study contributes information showing that MAT did not differ significantly among control, obese and type 2 diabetes subjects. It should be highlighted that the difference in IHL was significantly higher between the control and obese groups (253%) than that between the obese and type 2 diabetes groups (50%). The same pattern was observed when we calculated the variation of VAT between control and obese subjects (36%) and between obese and type 2 diabetes subjects (5.3%). Conversely, MAT showed a different and unique behavior, i.e., it was slightly lower in the obese group compared with control (11%) and was higher in type 2 diabetes subjects than that in the obese subjects (14.8%). These results clearly show that marrow adipocytes do not accumulate fat under conditions of energy surplus and adapt differently to nutritional variation. Indirectly, these results support previous clinical and experimental investigations showing that MAT increases under conditions of calorie restriction, whereas the other fat depots shrink (31). MAT in humans and mice exhibits the same pattern of adaptation under conditions of caloric restriction, with MAT expansion occurring in both species (31, 32, 33). Accumulated experimental evidence indicates that the incremental amount of MAT under caloric restriction results from progenitor recruitment and adipocyte differentiation rather than from adipocyte hypertrophy, arguing against a target for energy support in this process (34, 35). Intriguingly, mice and humans respond differently when exposed to a high-fat diet. The former exhibit a concomitant enhancement of WAT and MAT under a high-fat diet regimen (36), whereas the total amount of MAT remains unchanged in obese humans (37). Conceivably, the relatively smaller quantity of MAT in smaller animals such as mice accounts for trace level

changes in several physiological conditions. Further, human studies are necessary to investigate MAT quality and the profile of changes in bone adipocyte secretion during fat surplus.

A recent study on a group of non-diabetic participants predominantly consisting of normal and overweight women demonstrated a positive relationship between MAT and circulating glucose levels and showed no relationship between serum insulin levels or HOMA-IR and MAT (12). This study, which included normal weight, obese and type 2 diabetes individuals of both sexes, also supports these findings, demonstrating that MAT has a positive relationship with HbA1c values and no association with serum insulin levels or HOMA-IR. Moreover, an association was observed between VAT and IHL and the association was maintained after correction for age and BMI. In contraposition, no association was verified between MAT and VAT. The present results are not in line with those obtained by Bredella et  al. in a previous study evaluating obese women (38). The authors described a weak positive association between MAT and VAT in 47 healthy premenopausal women, whose BMI ranged from 18.1 to 41.4 kg/m2 with a mean age of 32.8 ± 7.1  years. Also, they found no association between age and MAT. The two studies are not directly comparable; this study also included diabetic patients, not only women but also men, and the mean age of the volunteers differed (38). Indirectly, the present results are supported by a recent study showing that higher HOMA-IR is associated with greater volumetric BMD and generally favorable bone microarchitecture, independent of body weight (39). Furthermore, the associations between HOMA-IR and bone microarchitecture persisted after adjusting for multiple potential covariates including time since menopause. Based on these results, the authors hypothesized that insulin resistance may protect, at least in part, against bone loss due to estrogen deficiency and/or aging in postmenopausal women (39).

Peptide secretion by WAT is largely influenced by the amount of lipids stored in adipocytes. Although leptin and proinflammatory adipokines increase with progressive triglyceride accumulation, adiponectin secretion is reduced during cell enlargement. As expected, leptin was slightly higher in the obese group, whereas adiponectin was higher in the control group, but there was no relationship between BMAT or BMD and leptin or adiponectin levels. A previous study reported a positive correlation between BMD and leptin, but the relationship disappeared after adjustment for fat mass (40). Experimental approaches indicate that

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leptin input through the hypothalamic ventromedial nucleus stimulates the sympathetic nervous system, hampering osteoblast activity (41). Conversely, there is also some evidence that leptin also exerts a direct stimulatory action on osteoblasts. In the clinical setting, another relevant factor accounting for difficulties in understanding the action of leptin on bone is the evolving process of leptin resistance with obesity. Pref-1, a peptide produced by adipocyte precursor cells, inhibits adipocyte and osteoblast differentiation. A previous study showed that patients with anorexia nervosa have higher or similar serum Pref-1 levels compared with age-matched healthy women (42). In this study, the circulating levels of Pref-1 were similar in control, obese and type 2 diabetic subjects. To our knowledge, only one study determined the serum levels of Pref-1 in these 3 groups (43). No significant difference was detected between control and obese subjects, but type 2 diabetes individuals had higher Pref-1 levels than control. It should be emphasized that the cited study was conducted solely on women with a mean BMI value compatible with severe obesity.

In anorexia nervosa, serum Pref-1 levels are increased and are positively correlated with MAT, whereas they are negatively correlated with BMD (42). More recently, it was shown that in women with anorexia nervosa, Pref-1 is responsive to either clinical recovery or transdermal estrogen therapy, which has an inhibitory effect on Pref-1 production (44, 45). Enhanced BMD was detected in both studies, and the first investigation showed a reduction in MAT (44). These relationships were not observed in the present normal weight, obese or type 2 diabetes subjects. Previous studies also indicate that estrogen deficiency is associated with MAT expansion (46); although estrogen levels were not measured, it can be predicted that estrogen did not account for the differences between the groups in relation to adipose tissue parameters. The women included were appropriately matched in relation to premenopausal and postmenopausal status: 36% and 64% in C group, 31% and 69% in O group and 27% and 73% in type 2 diabetes group respectively.

This study has some limitations. The sample size was small, and the study had a cross-sectional design. On the other hand, the groups of patients were well defined, and all diabetic patients had been diagnosed with the disease at least five years before the study. Also, no diabetic patient had nephropathy, clinical neuropathy or proliferative retinopathy. In addition, the methods used for fat assessment are accurate, allowing precise measurements of fat within different fat pads.

Conclusion

This study shows that marrow is neither a niche for fat accumulation in energy surplus nor has any association with insulin resistance. Our findings also suggest that glucose excess may be of pathophysiologic significance for the development of marrow adipose tissue.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis research was supported by FAPESP (grant 2012/14603-6), National Council for Scientific and Technological Development (CNPq) (grant 470279/2013-3), NAP-DIN (11.1.21625.01.0) and FAEPA, F J A P received financial support from the CNPq (307901/2014-9) and I M A received financial support from FAPESP (grant 2012/09527-9).

Author contribution statementI M A and F J A P conceived and designed the experiments; I M A, C E G S, A K N, M H N B, J E Jr and F J A P performed the experiments; I M A, C E G S, M H N B, J E Jr and F J A P analyzed the data; A K N did the statistical analysis; I M A and F J A P wrote the manuscript; C E G S, A K N, M H N B and J E Jr reviewed/edited the manuscript. Guarantor’s name: I M A and F J A P.

AcknowledgmentsThe authors are indebted to Marta T N Maibashi, Rita C Varrichio, Rodrigo Pessini, Luciana M Mijoler da Cunha for competent technical assistance and Elettra Greene for editorial assistance.

References 1 Despres JP & Lemieux I. Abdominal obesity and metabolic syndrome.

Nature 2006 444 881–887. (doi:10.1038/nature05488) 2 Premaor MO, Comim FV & Compston JE. Obesity and fractures.

Arquivos Brasileiros de Endocrinologia and Metabologia 2014 58 470–477. (doi:10.1590/0004-2730000003274)

3 Patsch JM, Li X, Baum T, Yap SP, Karampinos DC, Schwartz AV & Link TM. Bone marrow fat composition as a novel imaging biomarker in postmenopausal women with prevalent fragility fractures. Journal of Bone and Mineral Research 2013 28 1721–1728. (doi:10.1002/jbmr.1950)

4 Cutrim DM, Pereira FA, de Paula FJ & Foss MC. Lack of relationship between glycemic control and bone mineral density in type 2 diabetes mellitus. Brazilian Journal of Medical and Biological Research 2007 40 221–227. (doi:10.1590/S0100-879X2007000200008)

5 Prieto-Alhambra D, Premaor MO, Fina Aviles F, Hermosilla E, Martinez-Laguna D, Carbonell-Abella C, Nogues X, Compston JE & Diez-Perez A. The association between fracture and obesity is site-dependent: a population-based study in postmenopausal women. Journal of Bone and Mineral Research 2012 27 294–300. (doi:10.1002/jbmr.1466)

6 Nielson CM, Marshall LM, Adams AL, LeBlanc ES, Cawthon PM, Ensrud K, Stefanick ML, Barrett-Connor E & Orwoll ES. BMI and fracture risk in older men: the osteoporotic fractures in men study

Downloaded from Bioscientifica.com at 05/24/2021 04:41:38AMvia free access

PROOF ONLY

Euro

pea

n J

ou

rnal

of

End

ocr

ino

log

y176:1 29Clinical Study I M de Araújo and others Marrow adipose tissue in

type 2 diabetes

www.eje-online.org

(MrOS). Journal of Bone and Mineral Research 2011 26 496–502. (doi:10.1002/jbmr.235)

7 Schwartz AV. Epidemiology of fractures in type 2 diabetes. Bone 2016 82 2–8. (doi:10.1016/j.bone.2015.05.032)

8 Bonds DE, Larson JC, Schwartz AV, Strotmeyer ES, Robbins J, Rodriguez BL, Johnson KC & Margolis KL. Risk of fracture in women with type 2 diabetes: the Women’s Health Initiative Observational Study. Journal of Clinical Endocrinology and Metabolism 2006 91 3404–3410. (doi:10.1210/jc.2006-0614)

9 Premaor MO, Ensrud K, Lui L, Parker RA, Cauley J, Hillier TA, Cummings S & Compston JE. Risk factors for nonvertebral fracture in obese older women. Journal of Clinical Endocrinology and Metabolism 2011 96 2414–2421. (doi:10.1210/jc.2011-0076)

10 Saito M, Fujii K, Mori Y & Marumo K. Role of collagen enzymatic and glycation induced cross-links as a determinant of bone quality in spontaneously diabetic WBN/Kob rats. Osteoporosis International 2006 17 1514–1523. (doi:10.1007/s00198-006-0155-5)

11 Russell M, Mendes N, Miller KK, Rosen CJ, Lee H, Klibanski A & Misra M. Visceral fat is a negative predictor of bone density measures in obese adolescent girls. Journal of Clinical Endocrinology and Metabolism 2010 95 1247–1255. (doi:10.1210/jc.2009-1475)

12 de Paula FJ, de Araújo IM, Carvalho AL, Elias J, Salmon CE & Nogueira-Barbosa MH. The relationship of fat distribution and insulin resistance with lumbar spine bone mass in women. PLoS ONE 2015 10 e0129764. (doi:10.1371/journal.pone.0129764)

13 Tsatsoulis A, Mantzaris MD, Bellou S & Andrikoula M. Insulin resistance: an adaptive mechanism becomes maladaptive in the current environment – an evolutionary perspective. Metabolism 2013 62 622–633. (doi:10.1016/j.metabol.2012.11.004)

14 Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology 2012 142 711.e716–725.e716. (doi:10.1053/j.gastro.2012.02.003)

15 Cusi K. The role of adipose tissue and lipotoxicity in the pathogenesis of type 2 diabetes. Current Diabetes Reports 2010 10 306–315. (doi:10.1007/s11892-010-0122-6)

16 Leslie WD, Rubin MR, Schwartz AV & Kanis JA. Type 2 diabetes and bone. Journal of Bone and Mineral Research 2012 27 2231–2237. (doi:10.1002/jbmr.1759)

17 de Liefde II, van der Klift M, de Laet CE, van Daele PL, Hofman A & Pols HA. Bone mineral density and fracture risk in type-2 diabetes mellitus: the Rotterdam Study. Osteoporosis International 2005 16 1713–1720. (doi:10.1007/s00198-005-1909-1)

18 Vestergaard P. Discrepancies in bone mineral density and fracture risk in patients with type 1 and type 2 diabetes – a meta-analysis. Osteoporosis International 2007 18 427–444. (doi:10.1007/s00198-006-0253-4)

19 Bador KM, Wee LD, Halim SA, Fadi MF, Santhiran P, Rosli NF & Mustafa N. Serum osteocalcin in subjects with metabolic syndrome and central obesity. Diabetes and Metabolic Syndrome 2015 10 (Supplement 1) S42–S45. (doi:10.1016/j.dsx.2015.09.009)

20 Saber LM, Mahran HN, Baghdadi HH & Al Hawsawi ZM. Interrelationship between bone turnover markers, calciotropic hormones and leptin in obese Saudi children. European Review for Medical and Pharmacological Sciences 2015 19 4332–4343.

21 Iglesias P, Arrieta F, Pinera M, Botella-Carretero JI, Balsa JA, Zamarron I, Menacho M, Diez JJ, Munoz T & Vazquez C. Serum concentrations of osteocalcin, procollagen type 1 N-terminal propeptide and beta-CrossLaps in obese subjects with varying degrees of glucose tolerance. Clinical Endocrinology 2011 75 184–188. (doi:10.1111/j.1365-2265.2011.04035.x)

22 Rochefort GY, Rocher E, Aveline PC, Garnero P, Bab I, Chappard C, Jaffre C & Benhamou CL. Osteocalcin-insulin relationship in obese children: a role for the skeleton in energy metabolism. Clinical Endocrinology 2011 75 265–270. (doi:10.1111/j.1365-2265.2011.04031.x)

23 Lucey AJ, Paschos GK, Thorsdottir I, Martinez JA, Cashman KD & Kiely M. Young overweight and obese women with lower circulating osteocalcin concentrations exhibit higher insulin resistance and concentrations of C-reactive protein. Nutrition Research 2013 33 67–75. (doi:10.1016/j.nutres.2012.11.011)

24 Pittas AG, Harris SS, Eliades M, Stark P & Dawson-Hughes B. Association between serum osteocalcin and markers of metabolic phenotype. Journal of Clinical Endocrinology and Metabolism 2009 94 827–832. (doi:10.1210/jc.2008-1422)

25 Dobnig H, Piswanger-Solkner JC, Roth M, Obermayer-Pietsch B, Tiran A, Strele A, Maier E, Maritschnegg P, Sieberer C & Fahrleitner-Pammer A. Type 2 diabetes mellitus in nursing home patients: effects on bone turnover, bone mass, and fracture risk. Journal of Clinical Endocrinology and Metabolism 2006 91 3355–3363. (doi:10.1210/jc.2006-0460)

26 Oz SG, Guven GS, Kilicarslan A, Calik N, Beyazit Y & Sozen T. Evaluation of bone metabolism and bone mass in patients with type-2 diabetes mellitus. Journal of the National Medical Association 2006 98 1598–1604.

27 Weiss R. Fat distribution and storage: how much, where, and how? European Journal of Endocrinology 2007 157 (Supplement 1) S39–S45. (doi:10.1530/EJE-07-0125)

28 Moller JB, Pedersen M, Tanaka H, Ohsugi M, Overgaard RV, Lynge J, Almind K, Vasconcelos NM, Poulsen P, Keller C et al. Body composition is the main determinant for the difference in type 2 diabetes pathophysiology between Japanese and Caucasians. Diabetes Care 2014 37 796–804. (doi:10.2337/dc13-0598)

29 DeFronzo RA. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes 2009 58 773–795. (doi:10.2337/db09-9028)

30 Neeland IJ, Ayers CR, Rohatgi AK, Turer AT, Berry JD, Das SR, Vega GL, Khera A, McGuire DK, Grundy SM et al. Associations of visceral and abdominal subcutaneous adipose tissue with markers of cardiac and metabolic risk in obese adults. Obesity 2013 21 E439–E447. (doi:10.1002/oby.20135)

31 Bredella MA, Fazeli PK, Daley SM, Miller KK, Rosen CJ, Klibanski A & Torriani M. Marrow fat composition in anorexia nervosa. Bone 2014 66 199–204. (doi:10.1016/j.bone.2014.06.014)

32 Bredella MA, Fazeli PK, Miller KK, Misra M, Torriani M, Thomas BJ, Ghomi RH, Rosen CJ & Klibanski A. Increased bone marrow fat in anorexia nervosa. Journal of Clinical Endocrinology and Metabolism 2009 94 2129–2136. (doi:10.1210/jc.2008-2532)

33 Devlin MJ, Cloutier AM, Thomas NA, Panus DA, Lotinun S, Pinz I, Baron R, Rosen CJ & Bouxsein ML. Caloric restriction leads to high marrow adiposity and low bone mass in growing mice. Journal of Bone and Mineral Research 2010 25 2078–2088. (doi:10.1002/jbmr.82)

34 Cawthorn WP, Scheller EL, Learman BS, Parlee SD, Simon BR, Mori H, Ning X, Bree AJ, Schell B, Broome DT et al. Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction. Cell Metabolism 2014 20 368–375. (doi:10.1016/j.cmet.2014.06.003)

35 Cawthorn WP, Scheller EL, Parlee SD, Pham HA, Learman BS, Redshaw CM, Sulston RJ, Burr AA, Das AK, Simon BR et al. Expansion of bone marrow adipose tissue during caloric restriction is associated with increased circulating glucocorticoids and not with hypoleptinemia. Endocrinology 2016 157 508–521. (doi:10.1210/en.2015-1477)

36 Baum T, Yap SP, Karampinos DC, Nardo L, Kuo D, Burghardt AJ, Masharani UB, Schwartz AV, Li X & Link TM. Does vertebral bone marrow fat content correlate with abdominal adipose tissue, lumbar spine bone mineral density, and blood biomarkers in women with type 2 diabetes mellitus? Journal of Magnetic Resonance Imaging 2012 35 117–124. (doi:10.1002/jmri.22757)

37 Bredella MA, Gill CM, Gerweck AV, Landa MG, Kumar V, Daley SM, Torriani M & Miller KK. Ectopic and serum lipid levels are positively

Downloaded from Bioscientifica.com at 05/24/2021 04:41:38AMvia free access

PROOF ONLY

Euro

pea

n J

ou

rnal

of

End

ocr

ino

log

y176:1 30Clinical Study I M de Araújo and others Marrow adipose tissue in

type 2 diabetes

www.eje-online.org

associated with bone marrow fat in obesity. Radiology 2013 269 534–541. (doi:10.1148/radiol.13130375)

38 Bredella MA, Torriani M, Ghomi RH, Thomas BJ, Brick DJ, Gerweck AV, Rosen CJ, Klibanski A & Miller KK. Vertebral bone marrow fat is positively associated with visceral fat and inversely associated with IGF-1 in obese women. Obesity 2011 19 49–53. (doi:10.1038/oby.2010.106)

39 Shanbhogue VV, Finkelstein JS, Bouxsein ML & Yu EW. Association between insulin resistance and bone structure in nondiabetic postmenopausal women. Journal of Clinical Endocrinology and Metabolism 2016 101 3114–3122. (doi:10.1210/jc.2016-1726)

40 Thomas T, Burguera B, Melton LJ 3rd, Atkinson EJ, O’Fallon WM, Riggs BL & Khosla S. Role of serum leptin, insulin, and estrogen levels as potential mediators of the relationship between fat mass and bone mineral density in men versus women. Bone 2001 29 114–120. (doi:10.1016/S8756-3282(01)00487-2)

41 Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, Armstrong D, Ducy P & Karsenty G. Leptin regulates bone formation via the sympathetic nervous system. Cell 2002 111 305–317. (doi:10.1016/S0092-8674(02)01049-8)

42 Fazeli PK, Bredella MA, Misra M, Meenaghan E, Rosen CJ, Clemmons DR, Breggia A, Miller KK & Klibanski A. Preadipocyte factor-1 is associated with marrow adiposity and bone mineral density

in women with anorexia nervosa. Journal of Clinical Endocrinology and Metabolism 2010 95 407–413. (doi:10.1210/jc.2009-1152)

43 Kavalkova P, Touskova V, Roubicek T, Trachta P, Urbanova M, Drapalova J, Haluzikova D, Mraz M, Novak D, Matoulek M et al. Serum preadipocyte factor-1 concentrations in females with obesity and type 2 diabetes mellitus: the influence of very low calorie diet, acute hyperinsulinemia, and fenofibrate treatment. Hormone and Metabolic Research 2013 45 820–826. (doi:10.1055/s-0033-1353210)

44 Fazeli PK, Bredella MA, Freedman L, Thomas BJ, Breggia A, Meenaghan E, Rosen CJ & Klibanski A. Marrow fat and preadipocyte factor-1 levels decrease with recovery in women with anorexia nervosa. Journal of Bone and Mineral Research 2012 27 1864–1871. (doi:10.1002/jbmr.1640)

45 Faje AT, Fazeli PK, Katzman D, Miller KK, Breggia A, Rosen CJ, Mendes N, Misra M & Klibanski A. Inhibition of Pref-1 (preadipocyte factor 1) by oestradiol in adolescent girls with anorexia nervosa is associated with improvement in lumbar bone mineral density. Clinical Endocrinology 2013 79 326–332. (doi:10.1111/cen.12144)

46 Dang ZC, van Bezooijen RL, Karperien M, Papapoulos SE & Lowik CW. Exposure of KS483 cells to estrogen enhances osteogenesis and inhibits adipogenesis. Journal of Bone and Mineral Research 2002 17 394–405. (doi:10.1359/jbmr.2002.17.3.394)

Received 25 May 2016Revised version received 29 August 2016Accepted 4 September 2016

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