Fat and Sucrose Intake Induces Obesity-Related Bone Metabolism Disturbances: Kinetic and Reversibility Studies in Growing and Adult Rats.

JBMR

ORIGINAL ARTICLE

Fat and Sucrose Intake Induces Obesity-Related Bone Metabolism Disturbances: Kinetic and Reversibility Studies in Growing and Adult Rats C edric Lavet,1 Aline Martin,2 Marie-Therese Linossier,1 Arnaud Vanden Bossche,1 Norbert Laroche,1 Mireille Thomas,1 Maude Gerbaix,1 Patrick Ammann,4 Antoine Fraissenon,1 Marie-Helene Lafage-Proust,1 Daniel Courteix,3 and Laurence Vico1 Institut National de la Sant e et de la Recherche M edicale (INSERM) U1059, Laboratoire de Biologie integrative du Tissu Osseux, Lyon University, Saint-Etienne, France 2 Division of Nephrology, Center for Translational Metabolism and Health Feinberg School of Medicine, Northwestern University, Chicago, IL, USA 3 Laboratory of Metabolic Adaptations to Exercise in Physiological and Pathological conditions (AME2P, EA3533), Blaise Pascal University, Clermont University, Clermont Ferrand, France 4 Division of Bone Diseases, Department of Internal Medicine Specialties, Geneva University Hospital, Geneva, Switzerland 1

ABSTRACT Metabolic and bone effects were investigated in growing (G, n ¼ 45) and mature (M, n ¼ 45) rats fed a high-fat/high-sucrose diet (HFS) isocaloric to the chow diet of controls (C, n ¼ 30 per group). At week 19, a subset of 15 rats in each group (HFS or C, at both ages) was analyzed. Then one-half of the remaining 30 HFS rats in each groups continued HFS and one-half were shifted to C until week 27. Although no serum or bone marrow inflammation was seen, HFS increased visceral fat, serum leptin and insulin at week 19 and induced further alterations in lipid profile, serum adiponectin, and TGFb1, TIMP1, MMP2, and MMP9, suggesting a prediabetic phenotype and cardiovascular dysfunction at week 27 more pronounced in M than G. These events were associated with dramatic reduction of osteoclastic and osteoid surfaces with accelerated mineralizing surfaces in both HFS age groups. Mineral metabolism and its major regulators were disturbed, leading to hyperphosphatemia and hypocalcemia. These changes were associated with bone alterations in the weight-bearing tibia, not in the non-weight-bearing vertebra. Indeed in fat rats, tibia trabecular bone accrual increased in G whereas loss of trabecular bone in M was alleviated. At diaphysis cortical porosity increased in G and even more in M at week 27. After the diet switch, metabolic and bone cellular disturbances fully reversed in G, but not in M. Trabecular benefit of the obese was preserved in both age groups and in M the age-related bone loss was even lighter after the diet switch than in prolonged HFS. At the diaphysis, cortical porosity normalized in G but not in M. Hypocalcemia in G and M was irreversible. Thus, the mild metabolic syndrome induced by isocaloric HFS is able to alter bone cellular activities and mineral metabolism, reinforce trabecular bone, and affect cortical bone porosity in an irreversible manner in older rats. © 2015 American Society for Bone and Mineral Research. KEY WORDS: OBESITY; DIABETES; CALCIUM-PHOSPHATE METABOLISM; HISTOMORPHOMETRY; BONE STRENGTH

Introduction

A

ccumulating data suggest that obesity may be detrimental to bone.(1–4) Indeed, it does not simply increase mechanical loading on the skeleton: visceral fat causes adipocyte stress characterized by release of adipokines, infiltration by immune cells, and inflammatory signaling leading to type 2 diabetes and cardiovascular dysfunctions, which could respectively and differently affect bone health. However, obesity is not always associated with such abnormal metabolic features (ie, metabolically healthy obesity),(5) which complicates even more the understanding of obesity and

bone crosstalk. Yet the mechanisms behind the outcomes of obesity are not well understood, but dietary quality may modulate the obese metabolic phenotype(6–9) and therefore the related bone phenotype. In rodents, obesity has been induced by various high-fat diets whose physiopathological outcomes are similar to the human disease.(10,11) For example, Cao and colleagues(1,2) and Chen and colleagues(12) showed that growing rodents fed a hypercaloric high-fat diet had a lower bone mass in appendicular bones, associated with increased bone resorption and decreased bone formation, probably associated with a low-grade chronic inflammation (although not assessed in the study). However,

Received in original form January 26, 2015; revised form May 19, 2015; accepted June 29, 2015. Accepted manuscript online July 14, 2015. Address correspondence to: Laurence Vico, Ph.D, LBTO, Facult e de M edecine, 15 rue Ambroise Par e, 42023 Saint-Etienne cedex 2, France. E-mail: [email protected] Additional Supporting Information may be found in the online version of this article. Journal of Bone and Mineral Research, Vol. 31, No. 1, January 2016, pp 98–115 DOI: 10.1002/jbmr.2596 © 2015 American Society for Bone and Mineral Research

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in Europe and North America, diets not only provide too many calories but also excessive proportion of lipids along with a surplus of refined carbohydrates. Only a few studies linked nutritional behavior with bone health. Despite the lack of an absolute control, a long-term high-fat/high-sucrose (HFS) diet compared to low-fat/complex carbohydrates was found to impair geometrical and mechanical properties of tibial cortical bone in growing mice(13) and to alter femoral neck and vertebral morphology, bone mineral content (BMC), and mechanical properties in growing rats.(14–16) In contrast, in a transversal study in mature rats, Gerbaix and colleagues(17) found that a normocaloric HFS diet improved whole-body bone mineral density (BMD) (assessed by dual-energy X-ray absorptiometry [DXA]) while tibial and vertebral trabecular bone volume were maintained. Here, we aim to evaluate the long-term bone alterations in relation to metabolic disorders in a HFS diet-induced obesity in male rats as a function of age, because interactions between bone and obesity seem to be sex, age and site (weight or nonweight-bearing bone) specific.(18,19) In this context, our working hypothesis was that a qualitatively imbalanced diet may lead by itself to bone deterioration, which may be less reversible in adult than in younger animals. To test this, rats were fed a HFS diet with a high proportion of saturated fat and simple sugars, keeping the same calorie amount as the control rats fed a standard diet. This way, the study mimicked the poor quality but not the overfeeding of “junk food.” We investigated the impact of 19 and 27 weeks of HFS diet on general metabolism (body composition, glucose, and insulin plasma levels, lipid profile, inflammation, hepatic status, and serum adipokines) and bone parameters (hormones, bone cellular activities, bone marrow

adiposity and vascularity, and tissue microarchitecture and strength) in postweaning and adult rats. From 19 to 27 weeks, additional groups of HFS rats were switched to chow diet to evaluate the reversibility of metabolic and bone changes as a function of age.

Materials and Methods Animals and diets Seventy-five (75) 1-month-old (G: growing) and 75 6-month-old (M: mature) male Wistar rats supplied by Janvier (Le Genest St. Isle, France) were individually housed in a temperature- and humidity-controlled room. They were exposed to a reversed light-dark cycle with free access to water. In both age cohorts, animals were randomly assigned to a chow (C, n ¼ 30) or a HFS (n ¼ 45) diet. A subset of each group (n ¼ 15 C and 15 HFS) was killed by pentobarbital overdose after 19 weeks; ie, at 5.5 months of age for G rats (G-C and G-HFS) and 10.5 months for M rats (M-C and M-HFS). The rest of the population was maintained for 8 additional weeks and killed at 7.5 months (G rats) and 12.5 months (M rats). In both age cohorts, during the second period (week 19 to 27), the C group and 15 rats from the HFS group maintained the same diet (C¼>C and HFS¼>HFS); the other 15 HFS rats were shifted to the chow diet (HFS¼>C, Fig. 1). The protocol and procedures were in accordance with the European Community Standards on the care and use of laboratory animals (Ministere de l’Agriculture, France, Authorization 04827). The composition of diets is given in Table 1. The HFS diet was enriched in sucrose and lipids from animal origin. As compared to C, the HFS diet provided more saturated fatty acids (33%

Fig. 1. Experimental design. At the beginning of the experiment, 30 male Wistar rats fed with chow diet (30 C) and 45 with high fat sucrose diet (45 HFS) were included at both ages; ie, growing (left panel) and mature (right panel). Nineteen weeks later, 15 rats in each group (gray cells) were killed and processed for postmortem analyses (mCT, histomorphometry, serum testing). The other rats (white cells, 15 rats per group) remained under chow diet (C¼>C) or HFS diet (HFS¼>HFS), or switched from HFS to chow diet (HFS¼>C) for a period of 8 additional weeks. At week 27, all animals were killed. Ages of G and M rats were indicated at the beginning, after 19 and 27 weeks of the study (age in months, mo.). C ¼ chow diet; G ¼ growing; HFS ¼ high fat sucrose diet; M ¼ mature.

Journal of Bone and Mineral Research

OBESITY AND BONE METABOLISM IN YOUNG AND MATURE RATS

99

Lipid from plant (g)

– – – – – 35 35 – – 70 630

13

Lipid from animal (g)

– – – – – – – – 164 164 1476

31 17

204 – – – – – – – – 204 816

Protein (g)

Longitudinal assessments The following analyses were performed at week 19 and week 27 on all animals.

– – – 222 222 – – – – 444 1776 204 54 12 222 222 35 35 164 52 1000 4698

14 69

– 17

– – – – 670 2680

– – – – – 30 30 – – 600 540 – – – – – – – – – – – 170 – – – – – – – – 170 680 – – – 670

Casein Mineral mix Vitamin mix Cornstarch Sucrose Peanut oil Rapeseed oil Lard Cellulose Total (g) Energy (kcal/kg) % kcal

170 45 10 670 – 30 30 – 45 1000 3900

Protein (g) g/kg of diet Ingredients

Abdominal circumference and body composition (between vertebrae L1 and L5) were respectively assessed using a nonextendable measuring tape(20) and by DXA (Piximus; Lunar Corporation, Madison, WI, USA). “DXA- derived BMD and BMC were not considered in in vivo analysis because of their inaccuracy in obesogenic condition due to the important volume of fat and its spatial heterogeneity.(21–25) Micro–computed tomography of tibial trabecular compartment

Carbohydrate (g)

Lipid from animal (g)

38

Carbohydrate (g) g/kg of diet

Body morphometry

Lipid from plant (g)

High-fat high-sucrose diet (HFS) Chow diet (C) Table 1. Composition of the Experimental Diets

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versus 13%) and less monounsaturated fatty acids (49% versus 55%) and polyunsaturated fatty acids (18% versus 32%). In the HFS diet, cholesterol was provided by lard, resulting in an omega-6/omega-3 ratio higher than in standard diet (7.16 versus 5.63). During the protocol, we adjusted the daily amount of food to provide a similar mean intake of energy (C: 102 11 kcal, HFS: 109 15 kcal), protein, cellulose, vitamins, and minerals. Importantly, mean daily amounts of vitamin D (C: 65 7 UI, HFS: 70 9 UI), calcium (C: 186 20 mg, HFS: 198 27 mg), and phosphorus (C: 130 13 mg, HFS: 137 20 mg) were also similar.

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Micro–computed tomography (mCT) (VivaCT40; Scanco Medical, €ttisellen, Switzerland) was performed for all rats in vivo under Bru isoflurane anesthesia in the secondary spongiosa of the right tibia, at the start of the study and after 19 and 27 weeks. Data were acquired at 55 keV with a 10-mm cubic resolution. Threedimensional (3D) reconstructions of trabecular bone were generated using the following parameters: sigma: 1.2, support: 2, and threshold: 265 for M rats at each time course. For G rats, in which bone mineralization increased gradually during the experiment, the following parameters were used at weeks 0, 19, and 27, respectively: sigma: 1, 1, and 1.2; support: 2, 2, and 2; threshold: 216, 248, and 265. The structural parameters of trabecular bone: bone volume fraction (BV/TV, 1), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, 1/mm), trabecular separation (Tb.Sp, mm), structure model index (SMI, 1), connection density (Conn.D, 1/mm), degree of anisotropy (DA, 1), and trabecular bone mineral density (Tb.BMD, mg/cm3), were generated from a set of 110 slices starting below the growth plate. For BV/TV, Tb.N, Tb.Sp, and Tb.Th, results are presented as the difference between the values observed at week 0 and the time point of measurement.

Transversal assessments At the completion of experiments (week 19 or 27), the rats were fasted overnight and killed by intraperitoneal injection of pentobarbital. Sera, tibias, femurs, L2 vertebrae, perirenal and periepididymal fat pads, and liver were collected for the following experimental procedures. mCT of vertebral trabecular and cortical compartments L2 vertebrae (n ¼ 8/group) were scanned in 70% ethanol between the two growth plates. 3D reconstructions of trabecular bone were generated from the mid-distance between


growth plates to encompass 125 slices on each side of it. The same parameters as in the tibial trabecular compartment were assessed with the same segmentation parameters for G and M: sigma: 1.2, support: 2, threshold: 237. The vertebral cortical compartment was assessed from a pile of 200 slices similarly centered in the middle of the vertebra, with values of sigma: 0.8, support: 1, and threshold: 260.

tibias were then removed, decalcified, and imaged with mCT. Vessel volume per marrow volume (VV/MV), where MV is calculated as the total volume analyzed minus bone volume (obtained from mCT data acquired during the longitudinal study), was assessed with the following segmentation parameters: sigma: 1.2, support: 2, threshold: 290.(30) Liver histology

mCT of tibial cortical compartment Tibias (n ¼ 15/group) were scanned ex vivo, distally from the end of the tibial tuberosity, with a 10-mm cubic resolution at 55 keV. 3D reconstructions were generated from the first 50 slices using the following parameters: sigma: 0.8, support: 1, threshold: 316. Bone area and marrow area, as well as cortical mineral density, thickness, and porosity were calculated by integrating the value on each transverse section.

Hepatic steatosis was assessed in 10-mm-thick liver cryosections of frozen samples embedded in Neg-50 (Thermo Fisher Scientific, Belgium). The sections were fixed for 10 min in 3.7% formalin, rinsed with 60% isopropanol, and stained for 15 min with 0.5% Oil Red O (Sigma Aldrich) in water-diluted isopropanol (3:2). After washing with 60% isopropanol and counterstaining in Harris hematoxylin (Sigma Aldrich), the sections were mounted in Aquatex (Merck Millipore).

Bone histomorphometry

Biochemical assays

Bone labeling was performed by intraperitoneal injection of tetracycline (30 mg/kg of body weight; Sigma Aldrich, St. Louis, MO, USA) for 7 (for M groups) or 5 days (for G groups) and 1 day before killing. After killing, 8 right tibias and 8 L2 vertebrae per group (randomly selected) were embedded and processed as described in Pasqualini and colleagues.(26) The region of interest covered the tibial secondary spongiosa and the L2 vertebral body. According to standard nomenclature,(27) cellular parameters were determined: including osteoid surface (OS/BS, %), osteoid thickness (O.Th, mm), tartrate-resistant acid phosphatase (TRAcP)-positive osteoclastic surface (Oc.S/BS, %), mineral apposition rate (MAR, mm/day), single-labeled surface (sLS/BS, %), and double-labeled surface (dLS/BS, %), from which mineralizing surface per bone surface (MS/BS, %) and bone formation rate (BFR/BS, mm3/mm2/day) were derived. MS/OS (%) represents the ratio of mineralizing surface to osteoid surface and is equivalent to the fraction of the osteoid seam lifespan during which mineralization occurs. In addition, the relative volume of fat in the marrow cavity (Ad.V/MV, %) was measured on Goldnerstained sections using a manual counter and a 100-point grid.

Blood samples were collected by intracardiac puncture after killing. Sera were stored at –20°C until use. Biochemical assays were performed on 8 to 10 rats’ sera per group. Lipid profile (total cholesterol, high-density lipoprotein [HDL], and lowdensity lipoprotein [LDL] cholesterol, triglycerides [TG]; using the standard Trinder method), glucose, and alkaline phosphate (using hexokinase and para-nitrophenyl phosphate methods, respectively) were assessed by spectrophotometric measurements with an automated spectrophotometry analyzer (VISTA 1500; Siemens Healthcare Corporation, France). Serum phosphorus was determined with the ascorbic acid method as described in Chen and colleagues.(31) Serum calcium was measured by colorimetric assay (Biovision Inc, USA). Proinflammatory cytokines IL-1, IL-6, TNFa, and tissue inhibitor of metalloproteinase-1 (TIMP1) were measured by an antibody bead kit (Bio-plex Pro Assays; Bio-Rad Laboratories Inc., Hercules, CA, USA). An ELISA kit from the same manufacturer was used to assess vascular endothelial growth factor (VEGF). ELISA kits from USCN Life Science Inc (China) were used to measure insulin, Creactive proteins (CRPs), parathyroid hormone (PTH), and adiponectin. Intact fibroblast growth factor 23 (FGF23) was assessed with the Kainos ELISA kit (Kainos Laboratory Inc, Japan), whereas ELISA kits from ImmunoDiagnostic Systems (UK) were used to measure calcitriol, osteocalcin (OCN), procollagen crosslinks (CTX), and Tartrate-resistant acid phosphatase 5b (TRAcP5b). Serum undercarboxylated (GLU-OCN) and carboxylated OCN (GLA-OCN) were measured with enzyme immunoassay (EIA) kits from Takara (Otsu, Japan). Serum matrix metalloproteinase-2 and -9 (MMP2 and MMP9) were measured with ELISA kits from Biosource (USA).

Tibial macrostructural mechanical properties Three-point bending tests and proximal tibia axial compression tests were performed at week 27 as described in Amman and colleagues.(28) The mechanical resistance to failure was tested using a servo-controlled electromechanical system (Instron 1114; Instron Corp., High Wycombe, UK) with the actuator displaced at 2 mm/min, and both displacement and load were recorded. For the three-point bending test, the two-span distance was 20 mm and load was applied to the middle of the shaft. Load was applied to the tibial plate during the compression test. The maximal load (N), stiffness (slope of the linear part of the curve, representing the elastic deformation, N/ mm), and energy (area under the curve, N mm) were calculated. Vascular bed infusion and mCT evaluation of bone vascular network For contrasting of the bone vasculature, 7 randomly selected rats from each G and M groups were submitted to vascular injection of a barium sulfate solution (Micropaque solution; Guerbet, France) as described in Fei and colleagues.(29) The left


Quantitative analysis of gene expression by real-time PCR Femoral flushed marrow and marrow-free bone were collected and stored at –80°C. Tissues were homogenized in TRI reagent (Sigma-Aldrich) for quantitative RNA extraction. Nucleic acid pellets were processed with the RNeasy kit plus mini kit (Qiagen, Inc., Valencia, CA, USA). The purity and integrity of RNA were monitored with The Experion Automated Electrophoresis System (Bio-Rad Laboratories). Complementary DNA was synthesized using the iScript cDNA synthesis Kit (Bio-Rad Laboratories). Real-time PCR was performed using SYBR Green I dye (Light Cycler-FastStart DNA Master SYBR Green I;


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Diagnostics, Mannheim, Germany). Amplifications were performed with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) and data were analyzed with the CFX Manager software (Bio-Rad Laboratories). PCR primer sequences are listed in Supporting Table 1. Gene expressions were normalized using b-actin, cyclophilin A, and GAPDH for bone cDNA amplifications, and b-actin for marrow cDNA amplifications. Rats from the G-C groups were used as reference. Their gene expression levels were set as 1. RANKL and OPG mRNA were assessed, as well as expression of osteocyte markers: E11 (earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell), sclerostin (SOST), matrix extracellular phosphoglycoprotein (MEPE), Dentin matrix protein 1 (DMP1) and adipogenesis markers: leptin, PPAR-g, and fatty-acid-binding protein 4 (FABP4).

Statistical analyses The Gaussian distribution for each parameter was assessed by a Shapiro-Wilk test. In case of non-normal distribution, the data were log-transformed for analyses. Two-way analysis of variance (ANOVA), followed by a post hoc least significant difference (LSD) test, was used to examine the effects of age and diet on each parameter. Fat and lean (muscles and organs) mass along with bone and water mass are the main compounds of total body weight. In our model, weight variations were mainly due to increased abdominal fat mass. Pearson correlations were performed between bone mass or strength parameters and fat mass (in grams, assessed by DXA), and when significant, bone traits were adjusted with individual fat mass to appreciate whether they are commensurate to fat mass accumulation. Pearson correlations between bone mass or bone histomorphometric parameters and serum biochemistry were also performed at week 27. Analyses were carried out with the SPSS Advanced Statistics software (IBM Corp, Armonk, NY, USA) and data are presented as mean SE or as box plots in the figures. Values of p < 0.05 were considered significant.

Results Energy metabolism Because obesity could be associated with many metabolic disturbances that could differently affect bone metabolism, we first characterized the metabolic phenotype induced by HFS. At week 19, the HFS diet induced increased body weight, abdominal circumference, and visceral fat as assessed by DXA and weighing of the fat pad in G and M characteristic of visceral obesity (Fig. 2, Table 2A). Accumulation of perirenal and periepididymal fat was more than two-fold greater in G versus M. As expected, these changes were associated with a similar increase in serum leptin (Fig. 2). G-HFS and G-HFS¼>HFS livers also exhibited higher number and size of lipid droplets indicative of nonalcoholic steatosis (Supporting Fig. 1). Moreover, fasting insulin concentrations were higher in G-HFS (þ334%) and in M-HFS groups (þ86%), whereas fasting glucose was unchanged versus age-matched controls (Fig. 2B). These suggested the onset of insulin resistance.(32,33) At week 19, these metabolic disturbances were not associated with other abnormalities (Table 2A). Notably, adiponectemia was unaffected as reported in the “metabolically healthy profile” of obesity.(34–36) Further, the HFS diet did not induce inflammation at the systemic level and bone marrow level as assessed by the serum CRPs and inflammatory cytokines (IL-1b, IL-6, and TNFa),

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and their gene expressions in the bone marrow (Table 2A). The lipid profile (ie, serum HDL, LDL, and TG) was also not altered in the HFS groups (Table 2A). Only total cholesterol decreased by 20% in both age groups compared to controls, suggesting the onset of cardiovascular problems. At week 27, visceral adiposity also characterized HFS-fed rats. Body weight, visceral fat pad weight, and abdominal circumference were more important in G-HFS¼>HFS and M-HFS¼>HFS versus their respective controls (Fig. 2, Table 2B). But accumulation of perirenal fat pads was greater in G than in M as at week 19. In HFS rats, serum leptin was still increased in G-HFS¼>HFS (þ115%) with a tendency to increase in M-HFS¼>HFS (þ70%, p ¼ 0.096) versus their respective controls. Although the inflammation status was still unaltered by the HFS diet (Table 2B), adiponectin, which was unaffected at week 19, increased at week 27 in both G-HFS¼>HFS and M-HFS¼>HFS (þ70% to 75%). This supported new additional metabolic disturbances especially at the cardiovascular level.(37) Congruently, lipid profile as a marker of cardiovascular dysfunction(38,39) was altered. Although TG and LDL were still unchanged, total cholesterol and HDL decreased in rats of both ages. Of note, HDL tended to decrease more strongly in M rats than in G rats versus their respective controls (Table 2B). Furthermore, VEGF, MMPs, and their inhibitors (TIMPs), which are considered to be cardiovascular remodeling parameters, displayed significant alteration in HFS¼>HFS rats (Table 2B). As shown in hypertensive patients,(40,41) HFS-fed rats displayed a decrease of MMPs (MMP2 in M and MMP9 in G) and a respective increase and decrease of TIMP1 and VEGF (in M only) as compared to their respective controls. This suggested an increased vascular peripheral resistance and an elevated blood pressure in HFS-fed rats, but these alterations seems more important in M than in G. Finally, TGFb1 which displays a hypertrophic effects on cardiomyocytes(42) was also increased in G-HFS¼>HFS and M-HFS¼>HFS versus their respective controls and suggested a ventricular hypertrophy associated with hypertension. Knowing that insulin signaling in osteoblasts regulates glucose homeostasis by promoting OCN decarboxylation through osteoclast activity,(43,44) we analyzed the circulating forms either inactive OCN (carboxylate GLA) or active (GLUOCN). They remained unaltered at week 19. At week 27, two-way ANOVA detected a global diet effect (Table 2B), but post hoc analyses indicated that carboxylate GLA remained unchanged in G (p ¼ 0.097) and M (p ¼ 0.092) whereas GLU-OCN was decreased in both G-HFS¼>HFS and M-HFS¼>HFS as compared to their respective controls. Therefore, this could also reduce glucose tolerance and worsen the metabolic phenotype. In summary, HFS rats displayed visceral obesity associated with a prediabetic status and with gradual onset of metabolic disturbances, although no indices of systemic and osseous inflammations signs of cardiovascular dysfunctions appeared. Overall, at week 27 and despite less fat accumulation, these disturbances were more severe in M than in G rats at the end of the protocol. We then investigated whether this “mild” metabolic syndrome is able to impact phosphocalcic product and bone cellular activities.

Bone metabolism Phosphocalcic products Despite a persistent increase of serum calcitriol and an early increase of PTH in mature rats at week 19 (which then normalized at week 27), a progressive decline in calcemia


Fig. 2. (A) HFS diet induced visceral fat accumulation and metabolic disturbances. Abdominal fat mass was assessed by DXA, perirenal fat was weighted while leptin and adiponectin were measured in serum levels in growing and mature rats fed on chow ( ) or HFS ( ) at week 19 (top row) and week 27 (bottom row), and in HFS diet rats switched back to chow after week 19 ( ). (B) HFS diet induced prediabetic phenotype. Insulin and glucose levels were measured in growing and mature rats fed on chow ( ) or HFS ( ) at week 19. ANOVA p values are given in the graphs; Posttest p values ¼ age effect: $p < 0.05, $$p < 0.01, $$$p < 0.001, versus diet-matched growing; diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001, versus age-matched chow; switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001 versus age-matched HFS. n ¼ 13 to 15 rats/group for fat assessment and 6 to 8 rats/group for biochemical assays. HFS ¼ high fat sucrose diet; Aeff ¼ age effect, Deff ¼ diet effect, I ¼ interaction. Data are presented by box-and-whisker diagram with individual outliers plotted as open circles and extreme values as asterisks.

settled in HFS-fed rats (Fig. 3). Lower calcemia was first observed in M rats only, in which it began to decrease (–15% in M-HFS versus M-C rats, p ¼ 0.06), and then it became significant in HFS¼>HFS rats of both ages (–25% in M and –12% in G) at week 27. Despite this alteration in G at week 27, PTH and calcitriol remained unaltered in G. On the other hand, phosphatemia was found increased at week 27 in both G-HFS¼>HFS and M-HFS¼>HFS as compared to respective C¼>C but more importantly in G than in M (þ30% and þ15%, respectively), but serum FGF23, a major regulator of phosphate excretion, and its bone mRNA expression, remained


unaltered in G and M (Fig. 3, Supporting Table 2). In summary, mineral metabolism and its major regulators were disturbed under the HFS diet as shown by progressive hyperphosphatemia and hypocalcemia. Bone cellular activities and bone marrow features Major defects were observed in G and M in vertebra and tibia under the HFS diet at both weeks 19 and 27. They were characterized by dramatic drops in active osteoclasts (60%), osteoid surfaces (70%), and osteoid thickness (45%; data not


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G-C (mean SD)

Fat accumulation Weight (g)

B. After 27 weeks of diet

688 75

G-C¼>C (mean SD)

Fat accumulation Weight (g) 617 66 Abdominal fat percentage (%) 35.6 5.4 Waist circumference (mm) 217.9 15.7 Periepididymal mass (g) 19.7 3.3 S Abdominal fat pad mass (g) 40.6 7.9 Lipid profile Total cholesterol (mmol/L) 2.4 0.4 HDL (mmol/L) 1.3 0.1 LDL (mmol/L) 0.23 0.05 TG (mmol/L) 0.76 0.12 Inflammation status IL-1b (pg/mL) 306.3 39.9 IL-6 (pg/mL) 33.8 54.5 TNFa (pg/mL) 9.0 10.4 CRP (ng/mL) 249.1 15.3 Tibial bone marrow IL-1b mRNA (1) 1.0 0.20 Tibial bone marrow IL-6 mRNA (1) 1.0 0.10 Tibial bone marrow TN-a mRNA (1) 1.0 0.14 Growth factors and metalloproteinases MMP2 (ng/mL) 0.38 0.07 MMP9 (ng/mL) 0.74 0.41 TIMP1 (pg/mL) ND VEGF (pg/mL) 77.0 37.2 TGFb1 (pg/mL) 27493 19326 Bone turnover markers and metabolism regulators TRAPcP (UI) 0.980.58 CTX (ng/mL) 10.8 2.1 OCN (ng/mL) 201.7 60.7 Gla-OCN (ng/mL) 133.8 13.6 Glu-OCN (ng/mL) 32.38 5.0

A. After 19 weeks of diet

0.42 0.14 21730 59.8 42576

0.740.33 11.9 3.6 165.9 49.3 140.5 23.1 38.13 14.8

0.33 0.20 0.35 0.71 ND 65.2 55.4 46676 40273 1.490.29 11.3 2.3 231.8 63.6 147.9 20.9 39.12 10.1

776 67 ##

G-HFS¼>HFS (mean SD)

294.5 55.2 10.5 229.3

41.7 16.2 8.0 10.4 0.1 3 0.23 # 0.28 #

679 76.06 ¤¤¤

G-HFS¼>C (mean SD)

0.39 0.29 11795 31.1 3058

772 72.02 $$

M-C¼>C (mean SD)

Week 27

85.1 82.0 8.0 2.8 $ ND ND ND

0.4 0.1 $$$ 0.07 $$$ 0.41

263.6 12.9 11.1 247.8 0.82 0.75 0.74

2.1 0.8 0.25 0.57

0.2 # 0.2 0.07 0.21

54 $$$ 4$ 24.1 $$$ 4.1 10.1

2.0 1.2 0.22 0.74

66 ### 4.3 ### 20.7 ### 5.1 ### 10.5 ###

726 38.7 256.1 21.3 44.9

M-C (mean SD)

708 42.2 243.3 28.37 62.4

G-HFS (mean SD)

Week 19

828 53.45 $ #

M-HFS¼>HFS (mean SD)

0.067 0.107 0.107 0.048 0.141

0.405 0.657 0.210 0.460 0.850

0.698 0.688 0.490 0.630 0.099 0.018 0.049

0.002 0.605 0.187 0.923

0.000 0.000 0.000 0.000 0.000

Diet effect

0.274 0.249 0.624 0.529 0.178

0.252 0.134 N 0.197 0.068

0.124 0.890 0.777 0.401 – – –

0.881 0.971 0.377 0.674

0.003 0.245 0.020 0.016 0.019

Interaction

Age effect

0.000

Diet effect

0.328

Interaction

ANOVA (p values)

0.108 0.048 0.308 0.226 0.195

0.165 0.140 – 0.634 0.729

0.457 0.312 0.488 0.000 – – –

0.032 0.000 0.909 0.033

0.000 0.001 0.000 0.277 0.523

Age effect

ANOVA (p values)

704 81.45 # ¤¤¤ 0.002

M-HFS¼>C (mean SD)

0.69 0.48 16918 49.4 9215

66.2 96.1 8.2 9.3 $ ND ND ND

0.2 $ ## 0.2 $$$ 0.06 $$$ 0.42

66 $$$ ## 4.8 $ ### 14.7 $$$ ### 5$ 10.7 $ #

0.970.69 15.5 4.3 221.57 84.0 160.9 16.3 53.38 14.7

0.74 0.35 31552 102.1 26276

319.1 67.4 13.9 234.1

1.6 0.7 0.20 0.62

M-HFS (mean SD) 762 43.7 270.2 23.85 53.2

Table 2. Obesity Phenotype Induced by Isocaloric HFS Diet in Growing and Mature Rats After 19 (A) or 27 Weeks (B) of Diet



105

49.6 47.9 5.5 6.4 0.62 0.36 0.22

37.6 27.3 8.0 8.9 0.32 0.31 0.12

0.5 ¤ 0.4 ¤(.08) 0.12 0.22

0.71 17.2 164.9 110.5 24.4

0.61 18.14 46.5 17.7 3.9 #

0.33 21.5 176.0 129.3 31.3

0.29 7.9 42.9 18.0 6.3 ¤

332.2 78.5 5.9 6.4 0.32 0.37 0.14

0.5 0.4 0.08 $ 0.11 258.3 20.3 6.1 233.3 0.59 0.84 1.15

2.0 0.9 0.25 0.53

50.4 279.2 39.90 29.38 69.28

1.07 11.0 97.8 127.0 28.5

0.30 $$$ 2.1 51.8 $$ 7.6 $ 4.9

1.18 11.3 96.0 96.9 20.7

289.5 43.3 10.0 239.0 0.55 0.68 0.97

2.3 0.9 0.25 0.62

41.9 255.0 27.29 21.15 48.45

0.64 1.8 49.3 $$ 22.8 6.2 #

5149 #

0.19 $$ ¤ 0.50 $ 15503 32.5 ###

79.3 69.4 7.3 10.4 0.13 # 0.29 0.26

0.17 $$$ 2.1 $$$ 14.7 $$ 21.3 7.6 ¤

17273 8725 # 1.15 10.4 111.9 119.2 30.3

0.010 0.090 0.004 0.183 0.086

Age effect

0.000 0.036 0.000 0.015 0.152

0.712

0.000 0.000 – 0.678

0.347 0.282 0.169 0.256 0.140 0.188 0.320

0.315 0.535 0.664 0.010 0.005

0.021

0.138 0.024 0.030 0.002

0.510 0.883 0.603 0.398 0.042 0.179 0.955

0.006 0.000 0.885 0.963

0.000 0.000 0.000 0.000 0.000

Diet effect

0.484 0.441 0.965 0.746 0.746

0.141

0.047 0.217 – 0.004

0.285 0.689 0.397 0.886 0.583 0.684 0.329

0.638 0.056 0.867 0.485

0.498 0.388 0.051 0.384 0.054

Interaction

ANOVA (p values)

0.3 $ 0.007 0.1 $$$ ### 0.000 0.06 0.010 0.24 $ 0.007

5.9 ¤¤¤ 21.7 # ¤¤ 8.2 ¤¤¤ 4.7 # ¤¤¤ 11.3 # ¤¤¤

M-HFS¼>C (mean SD)

0.22 ### 0.77 0.50 0.36 29586 ## 30860 39.8 $$$ 40.7

113.9 11.5 4.3 12.9 0.11 # 0.20 0.46

0.4 # 0.2 $ ### 0.10 0.19 $

7.3 ### 16.9 ### 8.1 $$ # 5.3 # 15.2


0.38 $$$ 0.54 0.43 $$$ 0.46 10397 46801 36.3 $ 48.1 ### 9140 2349 18506

399.9 40.6 7.8 236.4 0.91 0.92 1.09

2.4 1.7 0.25 0.66

4.3 ¤¤¤ 40.3 4.4 $ 20.9 ### ¤ 239.0 24.6 12.9 ¤¤¤ 32.5 9.4 3.2 ¤¤¤ 25.4 5.4 15.1 ¤¤¤ 59.9 14.4

M-C¼>C (mean SD)

0.38 0.08 0.92 0.99 0.50 # 0.59 ND 16489 32.1 29.3 # ¤¤ 112.9

258.5 15.3 11.3 234.7 0.64 0.78 1.05

2.7 1.8 0.31 0.94

19427 11002 # 10156 6627 ¤

0.42 0.13 0.95 0.65 # ND 108.7 47.3

298.4 20.7 12.9 231.9 0.92 1.14 0.90

0.3 # 0.6 # 0.13 0.92

40.8 255.6 34.9 21.5 56.5

G-HFS¼>C (mean SD)

Week 27

ANOVA p values: Age effect: $p < 0.05, $$p < 0.01, $$$p < 0.001 versus diet-matched G; Diet effect and switching diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001 versus age-matched chow; Switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001 versus age-matched HFS. S ¼ sum of perirenal fat mass and periepididymal fat mass; HDL ¼ high-density lipoprotein; LDL ¼ low-density lipoprotein; TG ¼ triglycerides; IL-1b ¼ interleukin-1b; IL-6 ¼ interleukin-6; TNFa ¼ tumor necrosis factor a; CRP ¼ C-reactive protein; MMP2 ¼ matrix metalloproteinase-2; MMP9 ¼ matrix metalloproteinase-9; TIMP1 ¼ tissue inhibitor of metalloproteinase-1; VEGF ¼ vascular endothelial growth factor; TGFb1 ¼ transforming growth factor b1; TRAcP ¼ tartrate-resistant acid phosphatase; CTX ¼ carboxy-terminal collagen crosslinks; OCN ¼ osteocalcin; Gla-OCN ¼ carboxylated osteocalcin; Glu-OCN ¼ uncarboxylated osteocalcin; ND ¼ no data. Shading highlighted significantly altered data (ANOVA).

0.20 9.28 21.0 22.1 8.5

5426

0.37 0.10 1.69 0.49 ND 76.6 62.7

50.4 24.3 7.7 5.5 0.30 0.54 0.20

264.0 14.0 8.4 233.2 1.00 1.00 1.00

2.2 1.4 0.34 1.06

0.4 0.3 0.04 0.14

2.8 2.0 0.33 0.84

3.9 ### 11.3 ### 13.5 ### 5.3 ### 17.5 ###

4.6 22.8 9.2 4.3 12.2

36.6 225.3 32.5 22.2 54.8

46.1 270.9 55.0 27.9 82.9


G-C¼>C (mean SD)

TGFb1 (pg/mL) 12825 Bone turnover markers and metabolism regulators TRAcP (UI) 0.42 CTX (ng/mL) 12.9 OCN (ng/mL) 170.6 Gla-OCN (ng/mL) 134.6 Glu-OCN (ng/mL) 33.2

Abdominal fat percentage (%) Waist circumference (mm) Perirenal mass (g) Periepidymal mass (g) S abdominal fat pad mass (g) Lipid profile Total cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) TG (mmol/L) Inflammation status IL-1b (pg/mL) IL-6 (pg/mL) TNFa (pg/mL) CRP (ng/mL) Tibial bone marrow IL-1b mRNA (1) Tibial bone marrow IL6 mRNA (1) Tibial bone marrow TNFa mRNA (1) Growth factors and metalloproteinases MMP2 (ng/mL) MMP9 (ng/mL) TIMP1 (pg/mL) VEGF (pg/mL)

B. After 27 weeks of diet

Table 2. (Continued)

Fig. 3. HFS diet altered phosphate calcium metabolism on the long term. PTH, calcium, calcitriol, FGF23, and phosphate levels were measured in growing and mature rats fed on chow ( ) or HFS ( ) at week 19 (top row) and week 27 (bottom row), and in HFS diet rats switched back to chow after week 19 ( ). ANOVA p values are given in the graphs. Posttests p values ¼ age effect: $p < 0.05, $$p < 0.01, $$$p < 0.001, versus diet-matched growing; diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001, versus age-matched chow; switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001, versus age-matched HFS. n ¼ 6 to 8 rats/group for biochemical assays. HFS ¼ high fat sucrose diet; PTH ¼ parathyroid hormone, FGF23 ¼ fibroblast growth factor 23; Aeff ¼ age effect; Deff ¼ diet effect; I ¼ interaction. Data are presented by box-and-whisker diagram with individual outliers plotted as open circles and extreme values as asterisks.

shown) in both G and M (Fig. 4A, B). Additionally, in the tibia marrow OCN and TRAcP mRNA were also significantly decreased in M-HFS¼>HFS rats, and a similar trend was observed in GHFS¼>HFS at week 27, while RANKL and OPG mRNA (and their ratio) did not differ from controls, suggesting that bone cellular activities remained balanced (Supporting Table 2). Despite similar bone turnover alteration in weight-bearing and non-weight-bearing bones, bone formation rate appeared to be differently altered as a function of load. Indeed, in obese groups, BFR/BS in tibia was not reduced as compared to control (except slightly in growing obese rats at the end of the protocol). On the other hand, in vertebrae of obese rats BFR/BS dramatically fell at week 19 and its value remained so until week 27, whereas in lean control rats BFR/BS declined between week 19 and 27 to finally match the BFR/BS of obese rats. This suggested an age-dependent early decrease of bone formation rate in HFS groups (Fig. 4A). Surprisingly, at both tibia and vertebra, the ratio of mineralized surfaces per osteoid surfaces (MS/OS) was significantly increased in all HFS groups, suggesting that whatever the ages, osteoid mineralized more rapidly (Fig. 4A, B). To further document bone cellular activity changes we quantified serum levels of resorption markers and found that CTX and TRAcP were unaffected. Because osteocytes control bone remodeling, we checked tibia mRNA expression of osteocyte markers: E11, SOST, MEPE, and DMP1. None was changed by the HFS diet at week 27 (Supporting Table 2). These unaffected systemic markers of resorption and osteocyte protein expression suggested that bone cellular changes are restricted to trabecular compartments at least at the two time points analyzed. We then asked whether there are relationships between metabolic and bone cellular activities at week 27. Strong negative correlations were observed between osteoid or osteoclast parameters and adiponectin (not leptin) in M. In addition, in G

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and M rats, HDL positively correlated with osteoid and osteoclast parameters whereas TGFb1 negatively correlated with osteoid and osteoclast parameters only in M (Supporting Table 3). Marrow adipogenesis might be a critical event during obesity. However, at both weeks 19 and 27, tibial marrow fat was found to be unchanged under the HFS diet in G and M rats (data not shown), as was leptin marrow gene expression. Congruently, the expression of PPAR-g and FABP4, two markers of adipocyte differentiation, were also unaltered at week 27 in G and M (Supporting Table 2). Because the onset of diabetes could influence vascular architecture, we quantified blood vessels in the tibial metaphysis of mature rats. At week 19, VV/MV was unaltered by the HFS diet in G and M rats. In contrast, at week 27 VV/MV was decreased by 55% in M-HFS¼>HFS versus M-C¼>C rats, while it stayed unaltered in G rats (Fig. 4C). In summary, HFS-fed rats displayed a low but still coupled bone turnover associated with a rapid mineralization of the osteoid seam. In HFS groups, bone formation rate was preserved in weight-bearing bone but decreased rapidly at non-weightbearing sites. Marrow adiposity was unaffected by the HFS diet but the vascular network was impaired in M only.

Bone mass, geometry, and strength index Tibia During growth (ie, until week 19 in G), the trabecular bone acquisition at secondary spongiosa of the proximal metaphysis was greater (ie, from week 0 to week 19 in G), whereas during aging the trabecular bone loss was lower (ie, from week 0 to week 19 in M; Fig. 5) in HFS versus C. After adjustment by individual fat mass in G, where fat mass correlated with trabecular bone mass (r ¼ 0.24, p ¼ 0.007), differences between Journal of Bone and Mineral Research

Fig. 4. HFS diet dramatically reduced resorption and formation activities. Histomorphometric parameters were assessed at proximal tibia (A) and L2 vertebrae (B) within the trabecular compartment in growing and mature rats fed on chow ( ) or HFS ( ) at week 19 (top row) and week 27 (bottom row), and in HFS diet rats switched back to chow after week 19 ( ). (C) HFS diet decreased tibia vessel volume in mature rats. VV/MV was assessed in growing and mature rats fed on chow ( ) or HFS ( ) at week 19 and week 27, and in HFS diet rats switched back to chow after week 19 ( ) by mCT acquisition preceded by vessels contrasting by barium sulfate solution injection. ANOVA p values are given in the graphs. Posttest p values ¼ age effect: $p < 0.05, $ $p < 0.01, $$$p < 0.001, versus diet-matched growing; diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001, versus age-matched chow; switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001, versus age-matched HFS. n ¼ 8 rats/group. HFS ¼ high fat sucrose diet; OS/BS ¼ osteoid surface per bone surface; Oc.S/ BS ¼ osteoclastic surfaces per bone surface; BFR/BS ¼ bone formation rate per bone surface; MS/OS ¼ ratio of mineralizing surface to osteoid surface; VV/ MV ¼ vessel volume per marrow volume; Aeff ¼ Age effect, Deff ¼ Diet effect, I ¼ Interaction. Data are presented by box-and-whisker diagram with individual outliers plotted as open circles and extreme values as asterisks.



107

Fig. 5. HFS diet altered cortical and trabecular bone architectural parameters. Trabecular BV/TV, and cortical bone density and porosity were assessed by mCT in growing and mature rats fed on chow ( ) or HFS ( ) at week 19 (top row) and week 27 (bottom row), and in HFS diet rats switched back to chow after week 19 ( ). At week 19, DBV/TV was evaluated between week 0 and 19. At week 27, DBV/TV was evaluated between week 0 and 27. ANOVA p values are given in the graphs. Posttest p values ¼ age effect: $p < 0.05, $$p < 0.01, $$$p < 0.001, versus diet-matched growing; diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001, versus age-matched chow; switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001 versus age-matched HFS. n ¼ 13 to 15 rats/group. HFS ¼ high fat sucrose diet; BV/TV ¼ bone mass; Aeff ¼ age effect, Deff ¼ diet effect, I ¼ interaction. Data are presented by box-and-whisker diagram with individual outliers plotted as open circles and extreme values as asterisks.

HFS and C disappeared. These results suggested that in young animals, bone gain remains proportional to fat mass, whereas age-related bone loss is slowed down in fat mature animals. In addition, ultimate force and stiffness measured at week 27 did not change in G-HFS¼>HFS or M-HFS¼>HFS (Table 3). Cortical envelope response to the HFS diet differed from that of the trabecular compartment. Indeed, at the tibia cortex, during growth, BMD decreased in G at week 19, whereas cortical porosity increased in both G and M at week 27 (Table 3, Fig. 5) in HFS versus C without other alteration of structural parameters.

Lumbar vertebra During growth and during the aging-related bone loss period, cortical and trabecular bone were unchanged by the HFS diet (Table 3). Thus, the HFS diet led to different skeletal outcomes depending on the site investigated; ie, tibia or vertebra and the trabecular or cortical compartment.

Effects of switching back to chow diet Energy metabolism Even if waist circumference did not fully normalize in HFS¼>C in both G and M groups, perirenal, periepididymal, total visceral fat

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pad masses, and leptinemia normalized (Fig. 2, Table 2B). Moreover, in the liver of G-HFS¼>C, the number and size of lipid droplets decreased compared to G-HFS¼>HFS but did not fully normalize (Supporting Fig. 1). Adiponectin remained high in M, suggesting that recovery in mature rats was less easy than in younger animals (Fig. 2). Indeed, the HFS¼>C shift tended to normalize total cholesterol and HDL in G but not in M. Cardiovascular remodeling parameters and hypertensive markers were improved in HFS¼>C rats, but some drawbacks remained. In G-HFS¼>C, MMP9 was as low as in G-HFS¼>HFS, while TGFb1 normalized. In M-HFS¼>C, MMP2 and TIMP1 tended to normalize, while TGFb1 remained similar to MHFS¼>HFS (Table 2B). Finally, GLU-OCN levels were normalized in HFS¼>C at both ages. Although the recovery is not fully achieved, it is overall more advanced in G than in M rats. Bone metabolism Phosphocalcic products. Figure 3 indicates that when compared to rats who remained under HFS, switching from HFS to C was unable to reverse low calcemia, did not alter PTH level (as in the HFS diet), and kept serum calcitriol as high as in long-term HFS in M. Elevated phosphatemia seen in the HFS groups was reverted by the diet switch, and serum levels of FGF23 that did not change in HFS decreased in G (p ¼ 0.078, not significant [NS]) and in M (p < 0.05).




109

–0.08 0.008 $$$

0.05 0.005 ##

–1.62 0.093 $$$

2.96 0.16 ($)

2.49 0.15

–0.32 0.091 #

0.61 0.01 1295 6.7

0.010 $ 0.103 $ 0.005 5.6

0.59 0.01 1277 4.6 #

0.21 2.93 0.21 1043

M-C (mean SD)

assessments) 0.27 0.021 3.75 0.217 0.23 0.011 1033 5.8

G-HFS (mean SD)

Week 19


1066.96 6.12

0.20 0.006

0.009 5.43

0.19 0.019 $ 2.67 0.213 $

M-C¼>C (mean SD)

0.014 0.143

G-HFS¼>C (mean SD)

Vertebral trabecular and cortical compartments (transversal assessments) Trabecular BV/TV (1) 0.25 0.014 0.28 0.018 0.24 Trabecular Tb.N 3.34 0.101 3.70 0.115 3.46 (1/mm) Cortical thickness 0.21 0.006 0.22 0.007 0.22 (mm) Cortical density (mg 1043.88 4.27 1048.38 5.70 1052.33 HA/cm3) Tibial cortical compartment (transversal assessments)

G-C¼>C (mean SD)

B. Difference of Microarchitecture (mCT) and Biomechanical Parameters at Week 27 Week 27

Vertebral trabecular and cortical compartments (transversal Trabecular BV/TV (1) 0.25 0.017 Trabecular Tb.N (1/mm) 3.56 0.165 Cortical thickness (mm) 0.22 0.005 Cortical density (mg 1040 5.5 HA/cm3) Tibial cortical compartment (transversal assessments) Cortical thickness (mm) 0.58 0.01 Cortical density (mg 1293 3.9 HA/cm3) Cortical porosity (%) 2.39 0.17 Tibial trabecular compartment (longitudinal assessments) D BV/TV between 0.03 0.008 week 19 and week 0 (1) D Tb.N between –0.66 0.138 week 19 and week 0 (1/mm)

G-C (mean SD)

A. Difference of Microarchitecture (mCT) and Biomechanical Parameters at Week 19

Table 3. Difference of Microarchitecture and Biomechanical Parameters

0.010 0.129 ($) 0.006 6.4

1049.78 10.82

0.18 0.005 $$

0.21 0.018 $ 2.99 0.171 $$


1036.41 10.53

0.18 0.005 $$$

0.23 0.020 3.14 0.248

M-HFS¼>C (mean SD)

–1.14 0.035 $$$ ###

–0.05 0.002 $$ ##

2.76 0.09 ($)

0.61 0.01 1303 2.7 $$$

0.22 3.17 0.21 1027

M-HFS (mean SD)

0.000

0.000

0.949

0.687 0.345

0.320 0.220 0.641 0.077

0.464

0.609

0.318

0.694 0.017

0.778 0.737 0.178 0.467

0.690

0.000

0.004 0.001

Age effect

0.378

0.396

0.297 0.161

continued

0.058

0.077

0.451 0.611

Diet effect Interaction

ANOVA (p values)

0.000

0.000

0.002

0.090 0.005

0.013 0.001 0.104 0.788

Age Diet effect effect Interaction

ANOVA (p values)

110

LAVET ET AL.



0.03 0.007 2.55 0.201 $$$ 174 29 527 144

0.79 0.107 ## 178 34 373 179

0.14 0.017 $$$

0.05 0.007

0.03 0.007 ###

1.90 0.06 106.98 8.61 242.80 34.65

1293.05 4.24

1289.03 3.99 2.01 0.12 100.40 21.02 234.81 33.88

0.62 0.00

M-C¼>C (mean SD)

0.60 0.01

G-HFS¼>C (mean SD)

1.36 0.119 $$$ ### 173 34 493 63

0.02 0.005 $$

0.07 0.009 $$$ ###

2.69 0.21 $$ ### 117.52 14.00 283.05 45.15

1296.04 7.02

0.60 0.02


0.000

0.000 0.897 0.079

0.781

0.484

Age effect

1.30 0.058 $$$ ### 172 36 510 124

0.000 0.799 0.004

0.01 0.003 $$$ ¤ ## 0.000

0.05 0.003 $$$ ###

2.72 0.17 $$$ ### 99.502 13.81 245.52 34.70

1290.89 9.13

0.58 0.01

M-HFS¼>C (mean SD)

0.000 0.715 0.647

0.368

0.000

0.000 0.916 0.186

0.690

0.207

0.069 0.656 0.584

0.049

0.067

0.043 0.020 0.168

0.996

0.687

Diet effect Interaction

ANOVA (p values)

All parameters were assessed by mCT. Ultimate force and stiffness were evaluated by three-point bending test or metaphyseal compression test and microarchitecture parameters were assessed by mCT. ANOVA p values given in the table: Age effect: $p < 0.05, $$p < 0.01, $$$p < 0.001 versus diet-matched G; Diet effect and Switching diet effect: #p < 0.05, ##p < 0.01, ###p < 0.001 versus age-matched C; Switching effect: ¤p < 0.05, ¤¤p < 0.01, ¤¤¤p < 0.001 versus age-matched HFS. D BV/TV is characterized by bone acquisition in G at week 19 and by bone loss at week 19 in M and at week 27 in both G and M. BV/TV ¼ relative bone volume. Shading highlighted significantly altered data (ANOVA).

Cortical thickness 0.62 0.01 0.60 0.01 (mm) Cortical density (mg 1292 5.52 1294.06 4.32 HA/cm3) Cortical porosity (%) 1.84 0.06 2.16 0.12 # Ultimate force (N) 106.07 11.13 115.70 20.20 Stiffness (N/mm) 221.12 20.21 252.13 55.14 Tibial trabecular compartment (longitudinal assessments) D BV/TV week 27 to 0.01 0.009 0.01 0.009 # week 0 D BV/TV week 27 to 0.04 0.004 0.04 0.003 week 19 D Tb.N 1.50 0.166 1.00 0.187 # Ultimate force (N) 156. 37 176 50 Stiffness (N/mm) 358 59.6 440 157.1

G-C¼>C (mean SD)

Week 27

B. Difference of Microarchitecture (mCT) and Biomechanical Parameters at Week 27

Table 3. (Continued)

Bone cellular activities. As seen in Fig. 4, upon switching diet, bone cellular activities were in the process of recovery except for active osteoclastic surfaces (Oc.S/BS) in vertebrae of M-HFS¼>C that remained close to zero as in M-HFS. The recovery was fully or almost fully achieved for Oc.S/BS at tibia, and for osteoid surfaces (OS/BS) and osteoid thickness (not shown) at vertebra and tibia. Figure 4 also showed that the bone formation rate largely exceeded control values in both G and M after the diet switch, mainly because of higher mineralized surfaces (MS/BS, not shown). In tibia, gene expression of OCN and TRAcP (Supporting Table 2) were consistent with histomorphometry results, showing a restoration to control values after the diet switch. In summary, after switching to chow diet bone cellular activities recovered more efficiently in young than in mature rats and displayed a rebound in bone formation rate. Bone mass, geometry, and strength index. We evaluated the consequences of normalization (or even rebound) of the bone cellular activities on bone parameters after switching diets. In G, switching diets did not alter the trabecular benefit of the HFS diet (Table 3B, Fig. 5). In M rats who switched diets, trabecular bone loss was even more attenuated as compared to HFS¼>HFS (Table 3B, Fig. 5). In vertebra (Table 3B), no effects were observed. At the tibia cortex level, cortical porosity that was increased in G and even more in M under HFS, normalized only in G after switching to C, and stayed as high in M-HFS¼>C as in MHFS¼>HFS (Table 3B, Fig. 5). Cortical thickness, density, and ultimate force unaltered in both G and M under HFS did not change when rats were switched to C (Table 3B).

Discussion We evaluated to what extent bone health is compromised in overweight rats fed a poor quality, ie, food enriched in fat and sucrose, but normocaloric diet. Knowing that bone alterations could be age-specific,(18) we chose here to study two periods in the life of male rats: during growth (G, from weaning up to 5.5 months) and adult phase (G, from 5.5 months of age and M, from 6 to 10.5 or 12.5 months of age). Our data demonstrated that long-term exposure to HFS led to differential responses of cortical and trabecular bone dependent on bone localization. In the weight-bearing tibia metaphysis, HFS increased trabecular bone mass acquisition in young rats and attenuated trabecular bone loss in mature rats. Of note, the increase in BV/ TV in growing rats was proportional to fat gain. These positive effects were associated with dramatic and prolonged decrease in osteoclastic surfaces along with decreased osteoid seams at both vertebra and tibia in both G and M despite a worse metabolic phenotype in M. Further, because the RANKL/OPG bone marrow gene expression ratio was unchanged, it seems that bone cellular activities remained balanced, as already reported in mice under prolonged exposure to a high-fat diet(45) or in diabetic humans,(46–49) where low bone turnover might protect from bone loss due to aging. In these patients, a state of subclinical systemic inflammation and hyperglycemia is characteristically present: it is thus unlikely that inflammation (not seen at the systemic and bone marrow levels in the current study) is responsible for depressed resorption activity. Moreover, bone marrow vascularization was impaired in the tibia of HFS mature rats, similar to what was observed in the kidney of diabetic rats(50); this could also lead to decreased osteoclast and


osteoblast progenitor availability. Interestingly, we showed that MS/OS increased in all HFS groups, indicating that osteoid seams mineralized quicker than in controls. Because insulin, a bone anabolic factor,(51–53) increased in HFS, the osteoblast, that might express more insulin receptors,(54) should have an increased mineralizing activity. In addition, osteoclasts also express insulin receptors, whose stimulation results in the inhibition of resorption.(55) We also observed differences between bone formation at weight-bearing and non-weight-bearing sites. In obese rats, at the vertebrae, an age-dependent early decrease in bone formation occurred, while at the tibia BFR/BS was not reduced as compared to controls (except slightly in growing obese rats at the end of the protocol). Thus we can assume that load-induced obesity helps to maintain BFR/BS in weight-bearing bone while metabolic impairment has some negative influence on BFR/BS at non-weight-bearing sites. Finally, because resorption was similarly blunted in the HFS groups at both vertebrae and tibia, and despite vertebral bone formation decrease, trabecular bone could be maintained at the vertebrae and be reinforced at the tibias. At the cortical level, the adaptation to HFS differed from that in the trabecular compartment. Except an early and transient decrease in mineral density (at week 19) in young animals, no morphological or mineral density differences were seen at the tibia diaphysis. However, cortical porosity gradually increased in both growing and mature HFS rats. Studies using HRpQCT(46,47,56) also revealed an increased cortical porosity in obese humans. Thus, it seems plausible that obesity, with or without inflammation, decreased trabecular and increased cortical bone resorption with kinetics that remain to be defined, but associated, at least in our study, to unchanged serum level of TRAcP and CTX. In human studies, BMI value, which is most often used to diagnose obesity, does not reflect impairment in energy metabolism. Growing evidence indicates that features of metabolic syndrome have to be taken into account in the bone/fat axis (see Lecka-Czernik and Stechschulte(57) for review). Indeed, different high-fat diets (total enteral nutrition or hypercaloric diets) inducing multiple forms of obesity and probably different underlying mechanisms were mostly found to be detrimental,(1,2,12) but sometimes beneficial(58) or without effects(59) to bone mass in rodents. Gerbaix and colleagues,(17) using the same diet and model as in the present study, found a bone modulation similar to our results. To better understand the interplay between metabolic and osseous outcomes, we assessed their relationships. The metabolic profile of HFS rats is characterized by the onset of insulin resistance, with signs of cardiovascular malfunctions. These signs are more prominent in older rats although they have less visceral fat accumulation than young rats. Although leptin (and insulin), whose serum levels increased parallel to fat accumulation in HFS groups, have direct anabolic effects on bone,(60,61) it has been shown that high-sucrose diets induce leptin resistance.(62) Notably, we did not see any relationship between leptinemia and bone formation. Additionally, we found negative correlations between adiponectinemia and osteoid or osteoclast surfaces in mature rats that merit further investigations, because divergent results have been reported on the effects of adiponectin on bone cellular activities.(63–65) Studies in rodents report that osteoclasts are responsible for OCN decarboxylation, which in turn stimulates insulin secretion and adipocyte expression of adiponectin.(66) Here we do see that


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a low osteoclast number was associated to decreased undercarboxylated OCN, but we found that adiponectin increased. However, a low level of undercarboxylated form of OCN can contribute to worsened metabolic phenotype. Other serum parameters related to abdominal obesity could influence bone turnover. HDL protects mesenchymal stem cells from oxidative stress–induced apoptosis(67) and is correlated with serum OCN in human(68) and with osteoid and osteoclastic parameters in HFS rats at week 27. Adipose and serum levels of TGFb1 correlate with obesity in mice(69) and humans.(70,71) TGFb1 plays distinct roles at each stage of the osteoblast life cycle, inhibiting osteoblast matrix synthesis.(72) In the current study, TGFb1 increased and correlated negatively with histomorphometric bone cellular parameters in M-HFS¼>HFS, which had the worst metabolic phenotype. In humans, metabolic syndrome is associated with higher calcemia,(73,74) deficit in vitamin D,(75) and increased PTH.(76–78) However, such alterations might differ depending on insulin resistance or hyperglycemia severity.(79,80) In our model, HFS growing and mature rats displayed decreased calcemia and increased phosphatemia. One explanation for hypocalcemia might be a decrease in intestinal calcium absorption induced by the fructose generated by sucrose breakdown into glucose and fructose in the duodenum.(81) In mature rats, low serum calcium induced secondary hyperparathyroidism associated with a PTHrelated rise in serum calcitriol at week 19. Importantly, this early and transient increase in PTH might have been responsible for the increase in cortical porosity. Then at week 27, PTH levels normalized while serum calcium still declined despite higher calcitriol. The lack of long-term adequate PTH response was previously seen in diabetic patients with poor metabolic control during experimentally induced and maintained hypocalcemia,(82) probably because hyperinsulinemia is able to reduce PTH secretion.(83) In our model, osteoclast numbers are decreased and possibly osteoblast numbers (low OS/BS in HFS), suggesting a skeletal resistance to PTH. Furthermore, despite calcitriol increase, it failed to promote intestinal and renal calcium absorption. Contrarily to calcemia, which decreased, phosphatemia increased in M-HFS rats at week 27. In addition to calcitriol, PTH, and calcium, phosphate is mainly regulated by FGF23 produced predominantly by osteocytes.(84,85) Recent studies showed that FGF23 serum levels positively correlated with obesity, fat mass, and insulin resistance in obese adolescents.(86) Here, we did not observe any change in serum FGF23 or in other osteocyte markers (E-11, SOST, MEPE, and DMP-1, which controls FGF23 transcription) under the HFS diet. Thus we found that HFS deregulated calcemia and phosphatemia, even in the absence of severe metabolic syndrome. It is remarkable that the obesogenic effects of the HFS diet reversed almost completely after switching to chow diet in the absence of caloric restriction. Nonetheless, recovery in mature rats was less easy than in younger animals, with adiponectin, TGFb1, total cholesterol, and HDL failing to normalize despite the normalization of weight and abdominal fat. Calorierestrictive diets were most often used to induce weight loss, but were reported to concomitantly accelerate bone loss and sometimes increase risk of fracture.(87–89) In addition to decreased load due to weight loss, increased PTH, and reduced intestinal calcium absorption might be additional mechanisms responsible for bone loss.(90) In our study, despite the fact that diet calcium intake was similar in HFS and chow diet, and that PTH level did not change, calcemia remained

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low in both G and M rats after diet switching. Thus, even without calorie restriction, intestinal calcium absorption remains probably impaired despite a high calcitriol level. In addition, the active form of vitamin D remained elevated in mature rats under HFS. It has been recently reported that supplementation with vitamin D in obese postmenopausal women during weight loss failed to normalize calcium absorption, despite a rise in serum 25-hydroxyvitamin D, leaving the mechanism unexplained.(91) Furthermore, this persistent hypocalcemia was observed despite full (growing rats) or partial (mature rats) recovery of osteoclastic activities. This led us to think that the low bone remodeling occurring in HFS groups is not the main factor inducing hypocalcemia, even if a slow bone turnover certainly limits calcium normalization. In contrast to calcium, serum phosphate, which was elevated in young HFS animals, normalized upon diet switching along with a decrease in serum FGF23, significant in M rats. Intestinal uptake of phosphate occurs mainly through passive diffusion and saturable/facilitated absorption, suggesting that phosphatemia was mainly restored by normalization of bone turnover after diet switch. After diet switch, we observed a dramatic increase in bone formation rate, over the normal control values, due to increased mineralized surfaces (MS/BS; data not shown) and almost complete normalized osteoid surfaces at both ages. Further osteoclastic surfaces fully recovered in G but not in M, which could be related to the remaining metabolic drawback in M. This imbalance could lead to trabecular bone maintenance in G while age-related trabecular bone loss was even more alleviated in M who switched diet than in rats maintained on HFS. Therefore, in both G and M tibias, trabecular benefit of the obese phenotype was preserved. At the tibia diaphysis, the cortical porosity elevated in HFS normalized in young but not in mature rats.

Summary/Conclusion An isocaloric HFS diet induced abdominal obesity and a prediabetic condition with signs of cardiovascular dysfunction including lower medullary vascularization, without any systemic or bone marrow inflammation evidence. Most of these events were more severe in M rats than in G despite higher visceral fat accumulation in G. Despite a gradual worsening of the metabolic profile in HFS between weeks 19 and 27, bone cellular activities remained coupled but were dramatically and similarly impaired in G and M rats. Switching to chow diet normalized bone cellular activities despite some persistent metabolic drawback in M. Mineral metabolism is also disturbed as shown by hyperphosphatemia at week 27 in G and M rats, and persistent hypocalcemia even after diet switch. At the skeletal level, HFS appeared to be beneficial for trabecular appendicular bone mass, but a progressive cortical porosity settled in, while cortical thickness and density were not modulated as a function of fat mass. Weight loss due to normalization of diet macronutrients preserved the trabecular benefit of the obese phenotype in G and M and even decreased the trabecular bone loss in M. At the tibias cortical porosity remained elevated in M.

Disclosures All authors state that they have no conflicts of interest.


Acknowledgments This work was funded by the French Fondation pour la Recherche Medicale (FRM, contract number DVO20081013479), by the Institut National de la Sante et de la Recherche Medicale (INSERM), and the University of St-Etienne through basal funding to affiliated laboratories. We thank Anne Pelle, Geoffroy Marceau, Vincent Sapin, Clementine Mouty, Pierre Mouty, Isabelle Badoud-Georges, and Severine Clement for technical assistance, and the staff of the PLEXAN facility at University of St-Etienne for the care of the animals. Authors’ roles: Study design: CL, DC, MHLP and LV. Study conduct: CL, ML, AV, NL, MT, MG and AF. Data collection: CL, ML, AV, NL, MT, MG and AF. Data analysis: CL. Data interpretation: CL, AM, PA, MHLP and LV. Drafting manuscript: CL, AM and LV.

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Withdrawal of dietary phytoestrogens in adult male rats affects hypothalamic regulation of food intake, induces obesity and alters glucose metabolism.

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats.

Dietary high-fat lard intake induces thyroid dysfunction and abnormal morphology in rats.

high-sucrose diet diabetic mouse model.

Lymphocyte populations in mouse bone marrow: quantitative kinetic studies in young, pubertal and adult C3H mice.

The effects of nicotine self-administration and withdrawal on concurrently available chow and sucrose intake in adult male rats.

Reshaping faecal gut microbiota composition by the intake of trans-resveratrol and quercetin in high-fat sucrose diet-fed rats.

[Influence of date seed flour and cellulose on growth, food utilization and parameters of fat metabolism of growing and adult rats].

Transdifferentiation between bone and fat on bone metabolism.

Peak bone strength is influenced by calcium intake in growing rats.

Effect of diet on preference and intake of sucrose in obese prone and resistant rats.

Chronic alcohol intake induces reversible disturbances on cellular Na+ metabolism in humans: its relationship with changes in blood pressure.

Editorial: Oxytocin: Control of Bone and Fat Mass and Metabolism.

Postdischarge feeding of growing "preemies": concerns with limiting fat intake.

Ascites in broilers. 2. Disturbances in the hormonal regulation of metabolic rate and fat metabolism.

or energy expenditure: pathophysiological aspects.

Fructose and sucrose feeding during pregnancy and lactation in rats changes maternal and pup fuel metabolism.

Chronic nicotine induces a specific appetite for sucrose in rats.

High intake of sucrose and heart attacks.

Relaxin-3 mRNA levels in nucleus incertus correlate with alcohol and sucrose intake in rats.

High sodium intake during postnatal phases induces an increase in arterial blood pressure in adult rats.

Stress and Sucrose Intake Modulate Neuronal Activity in the Anterior Hypothalamic Area in Rats.

Prolonged high-fat diet induces gradual and fat depot-specific DNA methylation changes in adult mice.

High-fat diet preference in developing and adult rats.