176:1

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

Marrow adipose tissue spectrum in obesity and type 2 diabetes mellitus

Iana M de Araújo¹, Carlos E G Salmon², Andressa K Nahas³, Marcello H Nogueira-Barbosa¹, Jorge Elias Jr¹ and Francisco J A de Paula¹ Correspondence 1Department of Internal Medicine, Ribeirao Preto Medical School, 2Department of Physics, Faculty of should be addressed Philosophy, Sciences and Arts of Ribeirao Preto, and 3Department of Epidemiology, School of Public to F J A de Paula Health, USP, Ribeirão Preto, Sao Paulo,, Brazil Email [email protected]

Abstract

Objective: To assess the association of 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 allPROOF sites, BMD was negatively ONLY associated with osteocalcin. No association was observed between MAT and VAT. No relationship was observed among MAT and HOMA-IR, leptin, 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 European Journal European of Endocrinology not associated with VAT or insulin resistance. MAT is associated with glycated hemoglobin.

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

Introduction

Obesity and type 2 diabetes mellitus are part of a spectrum as visceral obesity, ectopic lipid deposition and an respectively linked as cause and consequence, embedded in altered profile in circulating levels of adipokines (1). a metabolic environment of insulin resistance generated by On the other hand, obesity and type 2 diabetes are adipose tissue and muscle. The performance of pancreatic β usually associated with normal bone mass, albeit this cells in insulin secretion determines the transition between feature does not necessarily mean that both have a normal glucose tolerance, pre-diabetes and type 2 diabetes, protective effect against fractures (2, 3, 4). Several a typical pathway followed by obese individuals. studies have shown that fracture risk is higher in obese Obesity and type 2 diabetes are also independent postmenopausal women (5) and obese men (6) and that risk factors for several disorders, including arterial fracture risk is significantly higher in type 2 diabetes (7, hypertension, dyslipidemia, macroangiopathy and 8). However, there are also data showing that, among nonalcoholic steatohepatitis. These disorders have obese individuals, fracture incidence is increased in mechanisms closely related to insulin resistance, such those with low bone mineral density (BMD) (9).

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

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10.1530/EJE-16-0448 Clinical Study I M de Araújo and others Marrow adipose tissue in 176:1 22 type 2 diabetes

The pathophysiology of bone disorder in obesity Subjects and methods and especially type 2 diabetes most likely includes specific factors that do not pertain to the universe of Subjects other chronic complications usually associated with Our study group comprised 24 controls (C: 14 females these metabolic diseases. and and 10 males), 26 obese subjects (O: 16 females and within the originate from the same 10 males) and 28 patients with type 2 diabetes . Several lines of evidence (15 females and 13 males). The clinical characteristics indicate that nutritional deprivation influences the of these groups are shown in Table 1. The study was commitment of progenitor cells toward differentiation approved by the Institutional Review Board of the into osteoblasts or adipocytes, whereas far less is known Ribeirao Preto Medical School, USP (#1149/2012), about the effect of energy surplus. and all subjects gave written informed consent to The mechanisms related to higher fracture risk participate. in obesity and type 2 diabetes have not been clearly Exclusion criteria were pregnancy, presence of a delineated. A previous study reported derangement of chronic disease known to affect bone metabolism, collagen crosslinking and cortical porosity as possible abnormal thyroid functioning, hypothalamic or pituitary mechanisms of decreased bone strength in type 2 disorders, use of estrogen/oral contraceptive, hormone diabetes (10). However, there is no consensus in the replacement therapy, glucocorticoid or literature about the impact of insulin resistance, adipose therapy (bisphosphonates, denosumab, teriparatide, tissue distribution and fat overflow to marrow adipose strontium ranelate and calcitonin). tissue (MAT) on BMD. Russel et al. reported that BMD Due to claustrophobia, 1 control, 1 obese and has an inverse relationship with the rate of visceral 5 type 2 diabetes subjects did not undergo any MRI adipose tissue (VAT) and subcutaneous adipose tissue examination. In addition, 3 individuals in the obese (SAT) in adolescent girls (11). There are also results group and 4 in the type 2 diabetes group refused to suggesting that bone volume is negatively associated complete the abdominal examination. Serum insulin with MAT and VAT in obese individuals. On the other levels were measured in 17 C and 17 O subjects, but hand, improvement in insulin sensitivityPROOF does not notONLY in type 2 diabetes individuals due to antidiabetic necessarily lead to a beneficial effect on bone mass. For therapy. instance, both calorie restriction and treatment with thiazolidinediones have a positive effect on insulin European Journal European of Endocrinology action and glucose tolerance, but concomitantly Methods provoke bone loss and MAT enhancement. The Biochemical assessment relationship between serum insulin levels and insulin resistance and different niches of fat depot, including Blood samples were collected between 0800 and 0900 h MAT, has been recently evaluated in non-diabetic after a 12-h overnight fast. Biochemical measurements women (12). The study showed an inverse relationship (calcium, glucose, glycated hemoglobin, phosphorous, between VAT and intrahepatic lipid (IHL) and serum albumin, alkaline phosphatase, aspartate aminotransferase levels of insulin, but no relationship was observed (AST), alanine aminotransferase (ALT) and creatinine) between MAT and circulating insulin levels. Despite were performed on the day of blood collection. Calcium, these lines of evidence, the effect of insulin resistance phosphorus, alkaline phosphatase, fasting glucose, and type 2 diabetes on bone quantity and quality has albumin, AST, ALT and creatinine were determined using not been assessed. an automatic biochemistry analyzer (Wiener lab, CT The focus of this study was to assess BMD and diverse 600 i, Thermo Fisher Scientific). The levels of glycated fat depots in obese and type 2 diabetes individuals and to hemoglobin (HbA1c) were measured by high-performance investigate the association of bone mass and MAT with liquid chromatography (D10 – Hemoglobin A1C Testing VAT, IHL and insulin resistance. The second objective System, Bio Rad). The serum aliquots for the other was to analyze the association of MAT with factors parameters were stored at −70°C until the day of the assay. originating in cell lines and osteoblasts (leptin, 25-Hydroxyvitamin D (25 (OH) D) (Liaison, DiaSorin, adiponectin, Pref-1 and osteocalcin) as well as with Saluggia VC, Italy), intact PTH (Immulite I, Siemens) metabolic control. and IGF-I (Immulite 2000 Siemens) were determined

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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 ± 8 Body mass index (kg/m²) 23.9 ± 1.7* 30.8 ± 4.0 33.0 ± 7.3 Height (m) 1.67 ± 0.1 1.64 ± 0.1 1.62 ± 0.1 Weight (kg) 67 ± 8* 83 ± 17 86 ± 16 Time of diagnostic (years) – – 11.8 ± 6.6 Glucose 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 ± 0.1 Phosphorus (mmol/L) 1.1 ± 0.2 1.1 ± 0.2 1.2 ± 0.3 PTH (pmol/L) 3.78 ± 1.86 4.28 ± 1.86 3.78 ± 2.33 Alkaline phosphatase (U/L) 165 ± 48 188 ± 55 180 ± 46 IGF-1 (µg/L) 143 ± 46 130 ± 60 130 ± 40 25(OH)D (mmol/L) 63 ± 23 56 ± 17 53 ± 20 Osteocalcin (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 ± 27.2 Pref-1 (ng/mL) 0.27 ± 0.15 0.28 ± 0.12 0.32 ± 0.18 L1–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 ± 4899 SAT (mm²) 20 753 ± 6721* 31 344 ± 12 594 28 544 ± 12 438 IHL (%) 2.9 ± 3.6* 7.5 ± 7.2 11.2 ± 8.8 MAT (%) 35.9 ± 9.3 31.8 ± 6.8 36.5 ± 8.4

BMD, bone mineral density; CTX, carboxy-terminal telopeptidePROOF of type I collagen; ONLY 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.

European Journal European of Endocrinology by chemiluminescence. The serum levels of osteocalcin voxel of 3.37 mL was positioned in the center of the (host-Easia Diasource, Louvain-la-Neuve, Belgium), third lumbar (L3) vertebral body. The point resolved Pref-1 (Quantikine Human Pref-1 R&D Systems), spectroscopy (PRESS) technique was applied using the leptin (Quidel, TECOmedical Group, Gewerbestrasse, following parameters: repetition time (TR) = 2000 ms, Sissach, Switzerland) and adiponectin (Millipore) were three echo times (TE) = 40/60/80 ms, 8 averages, without determined by enzyme immunoassay. The C-terminal fat or water suppression. telopeptide of type I collagen (CTX) was measured MRS data were processed with LCModel software by electrochemiluminescence (Cobas E 411, Roche (Version 6.1, http://www.s-provencher.com/pages/ Diagnostics). All intra- and inter-assay coefficients of lcmodel.shtml). The area values of the CH2 lipid peak at variation were lower than 10% and 20% respectively. 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/ 1H-MR spectroscopy of bone marrow in L3 (water + lipid) estimated from the corrected water and The volunteers underwent spine MRI in a 1.5 T system fat concentrations. (Philips ACHIEVA, Philips Medical Systems), as described previously (12). Abdominal magnetic resonance imaging Briefly, the subjects were positioned head first in the magnet bore in the prone position. A phased-array Abdominal images were acquired with a phased-array coil was positioned over the lumbar region. Sagittal torso coil. A coronal turbo-spin-echo (TSE) T2-weighted T2-weighted fast spin echo acquisition was used as a sequence with breath-holding was applied to localize the reference for the spectroscopy voxel placement. A single following scan volumes. Consecutively, a breath-holding

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axial gradient double-echo T1-weighted sequence, in phase SAS/STAT User’s Guide, Version 9.4, Cary, NC, USA: SAS (echo time = 4.2 ms) and out of phase (echo time = 2.1 ms, Institute INC., 2013). slice thickness = 6.0 mm), was acquired including the The linear regression model was applied, with age and whole abdomen, centered on the umbilical region. BMI considered as covariates. To determine the association The formula: fat = (SI in phase − SI out of phase)/ of the variables of interest, simple linear regression (2SI − in phase) was used to calculate IHL, VAT and SAT models (model 1) were adjusted by age and BMI (model from the averaged signal intensity (SI) in each region of 2), yielding the regression coefficients and R-square. All interest (ROI) or labels defined by image segmentation. analyses were carried out using the SAS 9.4 software. The The SI values in the previously mentioned formula refer level of significance was set at 0.05. to the pair in/out phase images. In the liver, a manual segmentation was performed to select four ROIs as Results representative segments of the liver at the level of the main portal vein. The label of the visceral and subcutaneous The study included 24 healthy controls, 26 obese subjects fat areas was defined using the Display software http://( and 28 type 2 diabetes patients. Table 1 shows the www.bic.mni.mcgill.ca/software/Display/Display.html) anthropometric characteristics and biochemical evaluation and a semiautomatic segmentation of an axial slice at of the subjects enrolled in this study. The O and type 2 the level of the umbilicus. This methodology has been diabetes groups were appropriately matched for age, sex, described previously in detail elsewhere (12). 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 Dual-energy X-ray absorptiometry 2 diabetes group. The three groups had similar serum levels Bone mineral density in the lumbar spine (L1–L4), total of calcium, phosphorus and alkaline phosphatase. hip and femoral neck was determined by dual-energy No significant differences in the serum levels of ALT, X-ray absorptiometry (Hologic Discovery Wi, QDR series, AST, creatinine, PTH, 25(OH)D and IGF-I were observed Waltham, MA, USA). The precision error was 1.2% for between groups (Supplements). The C and O groups L1–L4, 2.3% for the femoral neck and 2.7%PROOF for total hip. hadONLY similar serum levels of osteocalcin and CTX and BMD values are expressed as g/cm2. their values were higher than those observed in type 2 diabetes patients. BMD values for the lumbar spine (Fig. 1A), total hip

European Journal European of Endocrinology Statistical analysis (Fig. 1B) and femoral neck (Fig. 1C) were higher in type The data for the three groups were analyzed by one- 2 diabetes than those in O and C subjects (P < 0.05), but way ANOVA followed by the Duncan post-test. The did not differ between the O and C groups (Table 1). The confidence interval was 95%. ANOVA was calculated number of individuals with osteopenia (C = 11, O = 12, with the aid of the SAS 9.4 software (SAS Institute Inc., 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 relationship was observed between BMD and serum 58%, 57.6% and 32% in the C, O and type 2 diabetes insulin levels or HOMA-IR. A tendency to a negative groups respectively. association between BMD and MAT was observed in VAT was higher in type 2 diabetes than that in C the lumbar spine (P = 0.061), but this weak relationship subjects (Fig. 1E). Also, the type 2 diabetes and O groups was not maintained after adjustment for age and BMI. showed significantly higher IHL than the C group As expected, VAT and IHL exhibited a positive (P < 0.05; Fig. 1F). In contrast, no difference in MAT values association (Fig. 2A), which persisted after adjustment was found between the 3 groups (Table 1; Fig. 1F). for age and BMI. However, no association was observed Table 2 shows the results of linear regression between MAT and VAT (Fig. 2B) and VAT/SAT. Moreover, analysis. There was no association among lumbar in contrast to VAT, MAT exhibited no relationship with spine BMD and IHL, VAT or VAT/SAT rate. Also, no HOMA-IR (Fig. 2C) or serum insulin levels.

Table 2 Linear regression results.

Model 1 Model 2 Associations 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 0.16 Total hip BMD × IHL 0.003 0.19 −0.002 0.008 0.02 −0.001 0.73 −0.001 −0.001 0.18 Femoral neck BMD × IHL 0.004 0.15 −0.001 0.008 0.03 −0.001 0.55 −0.001 −0.001 0.26 L1–L4 BMD × VAT 2E−06 0.57 −1E−05 1E−05 0.01 −1E−06 0.7 −0.004 0.004 0.14 Total hip BMD × VAT 1E−05 0.07 −1E−06 1E−05 0.05 3E−06 0.43 −0.146 0.146 0.18 Femoral neck BMD × VAT 5E−06 0.21 −3E−06 1E−05 0.02 1E−06 0.88 −0.007 0.007 0.26 L1–L4 BMD × VAT/SAT −0.016 0.81 −0.154 0.121 0.01 0.023 0.73 0.018 0.028 0.14 Total hip BMD × VAT/SAT 0.067 0.33 −0.068 0.203 0.01 0.114 0.07 0.109 0.119 0.21 Femoral neck BMD × VAT/SAT −0.013 0.85 −0.15 0.125 0.01 0.039 0.53 0.034 0.044 0.26 L1–L4 BMD × insulin 0.0004 0.34 −5E−04 0.001 0.02 0.0001 0.91 −0.027 0.027 0.2 Total hip BMD × insulin 0.0004 0.36 −4E−04 0.001 0.02 0.0002 0.63 −0.125 0.125 0.1 Femoral neck BMD × insulin 0.0004 0.34 −5E−04 0.001 0.02 0.0001 0.91 −0.027 0.027 0.2 L1–L4 BMD × HOMA-IR 0.022 0.08 −0.002 0.047 0.08 0.017 0.27 0.016 0.018 0.1 Total hip BMD × HOMA-IR 0.019 PROOF0.08 −0.002 0.04ONLY0.09 0.013 0.3 0.012 0.014 0.16 Femoral neck BMD × HOMA-IR 0.018 0.14 −0.006 0.043 0.06 0.006 0.65 −0.116 0.128 0.22 L1–L4 BMD × MAT −0.004 0.06 −0.008 0 0.05 −0.002 0.26 −0.003 −0.002 0.13 Adiponectin × MAT −0.03 0.87 −0.34 0.29 0.01 −0.07 0.71 −0.42 0.29 0.05 Adiponectin × VAT −7E−04 0.01 −0.001 −2E−04 0.13 −6E−04 0.02 −0.001 −1E−04 0.15 European Journal European of Endocrinology Adiponectin × SAT 2E−05 0.83 −2E−04 0.0002 0.01 0.0006 <0.01 0.0002 0.001 0.21 Adiponectin × VAT/SAT −13.06 <0.01 −22.48 −3.64 0.14 −15.25 <0.01 −24.56 −5.95 0.23 Leptin × MAT −0.14 0.72 −0.93 0.65 0.01 0.17 0.68 −0.64 0.98 0.25 Leptin × VAT −7E−04 0.39 −0.002 0.001 0.01 −0.002 0.02 −0.003 −2E−04 0.32 Leptin × SAT 0.0015 <0.01 0.001 0.002 0.44 0.002 <0.01 0.001 0.0025 0.46 Leptin × VAT/SAT −48.08 <0.01 −71.67 −24.49 0.24 −40.35 <0.01 −62.27 −18.43 0.4 Pref-1 × MAT −0.003 0.18 −0.008 0.001 0.03 −0.004 0.12 −0.009 0.001 0.1 Pref-1 × IHL 0.001 0.81 −0.01 0.01 0.01 −0.001 0.79 −0.01 0.01 0.03 Pref-1 × VAT 1E−05 0.11 −1E−06 1E−05 0.04 5E−06 0.21 −3E−06 1E−05 0.06 Pref-1 × VAT/SAT 0.03 0.64 −0.11 0.17 0.01 0.05 0.49 −0.09 0.19 0.04 IHL × VAT 0.001 <0.01 0.0003 0.001 0.17 0.0005 0.011 −0.274 0.275 0.3 MAT × VAT 1E−05 0.955 −4E−04 0.0004 0.01 0.0002 0.395 −7.275 7.275 0.18 MAT × VAT/SAT 2.737 0.49 −4.999 10.473 0.01 0.667 0.85 −6.608 7.942 0.18 MAT × HOMA-IR −0.055 0.16 −0.131 0.021 0.05 −0.008 0.83 −0.084 0.067 0.27 MAT × insulin −0.857 0.34 −2.616 0.903 0.02 0.351 0.68 −1.342 2.043 0.31 L1–L4 BMD × osteocalcin −0.012 <0.01 −0.019 −0.006 0.17 −0.011 <0.01 −0.015 −0.006 0.22 Total hip BMD × osteocalcin −0.012 <0.01 −0.018 −0.005 0.15 −0.01 <0.01 −0.014 −0.005 0.21 Femoral neck BMD × osteocalcin −0.013 <0.01 −0.02 −0.006 0.16 −0.011 <0.01 −0.015 −0.006 0.26 Insulin × osteocalcin 0.751 0.58 −1.913 3.414 0.01 0.889 0.45 −1.397 3.175 0.31 Osteocalcin × glucose −0.28 0.09 −0.6 0.05 0.04 −0.22 0.2 −0.55 0.11 0.08 MAT × glucose 0.033 0.5 −0.063 0.129 0.01 0.057 0.28 −0.046 0.159 0.08 MAT × 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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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.

There were significant differences in the behavior of or insulin resistance and (c) is positively associated with circulating levels of adipocyte-originated factors among HbA1c. the 3 groups. Although adiponectin was higher in C than Several studies have observed that bone mass that in O and type 2 diabetes subjects, the serum levels of seems not to be negatively affected by type 2 diabetes leptin tended to be lower in the C group (P = 0.064). The and hyperglycemia (16, 17, 18). This study reaffirms 3 groups showed similar values of Pref-1. These 3 peptides these data and provides some additional interesting produced by adipocyte cell lines showed no association information. Bone mass was higher in type 2 diabetes with MAT (Table 2). not only compared with the control group but also A negative association was detected among compared with the obese group. These results indicate osteocalcin and lumbar spine (Fig. 2D), total hip (Fig. 2E) that BMD in type 2 diabetes individuals results from a and femoral neck (Fig. 2F) BMD. However,PROOF no association moreONLY complex configuration than simply the mechanical was observed between the serum levels of osteocalcin and and endocrine/paracrine influence of the adipose tissue insulin or glucose. existing in obese non-diabetic individuals. For instance, MAT showed no association with glucose, but was non-severely obese individuals do not share a low bone

European Journal European of Endocrinology positively associated with HbA1c. turnover profile with type 2 diabetes patients (19, 20, 21). Rochefort et al. (22) evaluated the serum concentrations Discussion of osteocalcin and insulin in a population of prepubertal obese and control children and observed that the Obesity reflects the high capacity of circulating levels of osteocalcin were similar in the two (WAT) to store lipids. However, the overflow of lipids groups. Similarly, another study conducted on non- from WAT to other tissues reveals that a certain limit diabetic overweight and obese adults reported that BMI exists concerning the ability of WAT to retain lipids in and body weight did not differ across tertiles of total or its domain. Excessive accumulation of fat within and undercarboxylated osteocalcin (23). On the other hand, subsequently outside adipose tissue leads to functional the present results seem to conflict with those obtained (insulin resistance) and structural disorders not only in in a previous study performed by Pittas et al. in a cohort adipose tissue itself but also in muscle, liver and pancreas study of individuals aged 65 years and older, showing that (13). Type 2 diabetes and nonalcoholic steatohepatitis osteocalcin is inversely associated with BMI and fat mass (NASH) are illustrative consequences of lipotoxicity (24). There are several differences between this study and triggered by the overflow of lipids (14, 15). Bone disease the one by Pittas et al. which render them incompatible in type 2 diabetes remains as a conundrum in which, for comparison: the age difference between the two unexpectedly, the role of MAT and insulin resistance has groups studied and, more importantly, the inclusion of scarcely been investigated. This study suggests that MAT some obese subjects who also had diabetes in the Pittas represents a diverse and sole type of adipose tissue: (a) it is study. The present results agree with those reported not a site for fat storage during energy surplus, (b) it seems in most studies of bone remodeling in type 2 diabetes, to have no relationship with VAT, serum levels of insulin which identified low bone formation and resorption

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activity based on the determination of biochemical changes in several physiological conditions. Further, markers (25, 26). Obese patients exhibited osteocalcin human studies are necessary to investigate MAT quality and CTX values similar to control. Moreover, there was a and the profile of changes in bone adipocyte secretion negative association between serum levels of osteocalcin during fat surplus. and lumbar spine BMD, suggesting that the high BMD A recent study on a group of non-diabetic participants in type 2 diabetes is closely related to low bone turnover. predominantly consisting of normal and overweight Although obesity is a major determinant of type women demonstrated a positive relationship between 2 diabetes mellitus, the site of fat allocation is more MAT and circulating glucose levels and showed no important than the amount of fat (27). Several steps are relationship between serum insulin levels or HOMA-IR involved in the onset of insulin resistance, especially and MAT (12). This study, which included normal the accumulation of fat in VAT and subsequently its weight, obese and type 2 diabetes individuals of both redistribution to pancreas, muscle and liver (28). Next, sexes, also supports these findings, demonstrating that β cell dysfunction becomes the last step in the onset of MAT has a positive relationship with HbA1c values and type 2 diabetes (29). Our data confirm previous studies no association with serum insulin levels or HOMA-IR. showing that VAT and IHL are significantly increased Moreover, an association was observed between VAT and in obese individuals (30) and even more so in type 2 IHL and the association was maintained after correction diabetes patients (15). This study contributes information for age and BMI. In contraposition, no association was showing that MAT did not differ significantly among verified between MAT and VAT. The present results control, obese and type 2 diabetes subjects. It should be are not in line with those obtained by Bredella et al. highlighted that the difference in IHL was significantly in a previous study evaluating obese women (38). The higher between the control and obese groups (253%) than authors described a weak positive association between that between the obese and type 2 diabetes groups (50%). MAT and VAT in 47 healthy premenopausal women, The same pattern was observed when we calculated the whose BMI ranged from 18.1 to 41.4 kg/m2 with a mean variation of VAT between control and obese subjects age of 32.8 ± 7.1 years. Also, they found no association (36%) and between obese and type 2 diabetes subjects between age and MAT. The two studies are not directly (5.3%). Conversely, MAT showed a differentPROOF and unique comparable;ONLY this study also included diabetic patients, behavior, i.e., it was slightly lower in the obese group not only women but also men, and the mean age of the compared with control (11%) and was higher in type 2 volunteers differed (38). Indirectly, the present results diabetes subjects than that in the obese subjects (14.8%). are supported by a recent study showing that higher European Journal European of Endocrinology These results clearly show that marrow adipocytes do HOMA-IR is associated with greater volumetric BMD and not accumulate fat under conditions of energy surplus generally favorable bone microarchitecture, independent and adapt differently to nutritional variation. Indirectly, of body weight (39). Furthermore, the associations these results support previous clinical and experimental between HOMA-IR and bone microarchitecture persisted investigations showing that MAT increases under after adjusting for multiple potential covariates including conditions of calorie restriction, whereas the other fat time since menopause. Based on these results, the authors depots shrink (31). MAT in humans and mice exhibits hypothesized that insulin resistance may protect, at least the same pattern of adaptation under conditions of in part, against bone loss due to estrogen deficiency and/ caloric restriction, with MAT expansion occurring in both or aging in postmenopausal women (39). species (31, 32, 33). Accumulated experimental evidence Peptide secretion by WAT is largely influenced by indicates that the incremental amount of MAT under the amount of lipids stored in adipocytes. Although caloric restriction results from progenitor recruitment leptin and proinflammatory adipokines increase with and adipocyte differentiation rather than from adipocyte progressive triglyceride accumulation, adiponectin hypertrophy, arguing against a target for energy support secretion is reduced during cell enlargement. As in this process (34, 35). Intriguingly, mice and humans expected, leptin was slightly higher in the obese group, respond differently when exposed to a high-fat diet. The whereas adiponectin was higher in the control group, former exhibit a concomitant enhancement of WAT and but there was no relationship between BMAT or BMD MAT under a high-fat diet regimen (36), whereas the total and leptin or adiponectin levels. A previous study amount of MAT remains unchanged in obese humans reported a positive correlation between BMD and leptin, (37). Conceivably, the relatively smaller quantity of MAT but the relationship disappeared after adjustment for in smaller animals such as mice accounts for trace level fat mass (40). Experimental approaches indicate that

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leptin input through the hypothalamic ventromedial Conclusion nucleus stimulates the sympathetic nervous system, hampering activity (41). Conversely, there This study shows that marrow is neither a niche for fat is also some evidence that leptin also exerts a direct accumulation in energy surplus nor has any association stimulatory action on osteoblasts. In the clinical setting, with insulin resistance. Our findings also suggest that another relevant factor accounting for difficulties glucose excess may be of pathophysiologic significance in understanding the action of leptin on bone is the for the development of marrow adipose tissue. evolving process of leptin resistance with obesity. Pref- 1, a peptide produced by adipocyte precursor cells, Declaration of interest inhibits adipocyte and osteoblast differentiation. A The authors declare that there is no conflict of interest that could be previous study showed that patients with anorexia perceived as prejudicing the impartiality of the research reported. nervosa have higher or similar serum Pref-1 levels compared with age-matched healthy women (42). In Funding this study, the circulating levels of Pref-1 were similar This research was supported by FAPESP (grant 2012/14603-6), National in control, obese and type 2 diabetic subjects. To our Council for Scientific and Technological Development (CNPq) (grant knowledge, only one study determined the serum 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 levels of Pref-1 in these 3 groups (43). No significant financial support from FAPESP (grant 2012/09527-9). 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 Author contribution statement the cited study was conducted solely on women with a I 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, mean BMI value compatible with severe obesity. M H N B, J E Jr and F J A P analyzed the data; A K N did the statistical In , serum Pref-1 levels are increased analysis; I M A and F J A P wrote the manuscript; C E G S, A K N, M H N B and are positively correlated with MAT, whereas they are and J E Jr reviewed/edited the manuscript. Guarantor’s name: I M A and F J A P. negatively correlated with BMD (42). More recently, it was shown that in women with anorexiaPROOF nervosa, Pref-1 ONLY is responsive to either clinical recovery or transdermal Acknowledgments estrogen therapy, which has an inhibitory effect on Pref-1 The authors are indebted to Marta T N Maibashi, Rita C Varrichio, Rodrigo Pessini, Luciana M Mijoler da Cunha for competent technical assistance production (44, 45). Enhanced BMD was detected in both and Elettra Greene for editorial assistance. European Journal European of Endocrinology 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. References Previous studies also indicate that estrogen deficiency is 1 Despres JP & Lemieux I. and metabolic syndrome. associated with MAT expansion (46); although estrogen Nature 2006 444 881–887. (doi:10.1038/nature05488) levels were not measured, it can be predicted that 2 Premaor MO, Comim FV & Compston JE. 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Received 25 May 2016 Revised version received 29 August 2016 Accepted 4 September 2016

PROOF ONLY European Journal European of Endocrinology

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