Morbid obesity attenuates the skeletal abnormalities associated with leptin deficiency in mice

in Journal of Endocrinology

Leptin-deficient ob/ob mice are morbidly obese and exhibit low total bone mass and mild osteopetrosis. In order to disassociate the skeletal effects of leptin deficiency from those associated with morbid obesity, we evaluated bone mass, architecture, gene expression, and indices of bone turnover in WT mice, ob/ob mice allowed to feed ad libitum (ob/ob), and ob/ob mice pair-fed equivalent to WT mice (pair-fed ob/ob). Mice were maintained at 32 °C (thermoneutral) from 6 to 18 weeks of age to minimize differences in resting energy expenditure. ob/ob mice were heavier, had more abdominal white adipose tissue (WAT), and were hyperglycemic compared with WT mice. Femur length, bone mineral content (BMC) and bone mineral density, and midshaft femur cortical thickness were lower in ob/ob mice than in WT mice. Cancellous bone volume (BV) fraction was higher but indices of bone formation and resorption were lower in ob/ob mice compared with WT mice; reduced bone resorption in ob/ob mice resulted in pathological retention of calcified cartilage. Pair-fed ob/ob mice were lighter and had lower WAT, uterine weight, and serum glucose than ob/ob mice. Similarly, femoral length, BMC, and cortical thickness were lower in pair-fed ob/ob mice compared with ob/ob mice, as were indices of cancellous bone formation and resorption. In contrast, bone marrow adiposity, calcified cartilage, and cancellous BV fraction were higher at one or more cancellous sites in pair-fed ob/ob mice compared with ob/ob mice. These findings indicate that the skeletal abnormalities caused by leptin deficiency are markedly attenuated in morbidly obese ob/ob mice.

Abstract

Leptin-deficient ob/ob mice are morbidly obese and exhibit low total bone mass and mild osteopetrosis. In order to disassociate the skeletal effects of leptin deficiency from those associated with morbid obesity, we evaluated bone mass, architecture, gene expression, and indices of bone turnover in WT mice, ob/ob mice allowed to feed ad libitum (ob/ob), and ob/ob mice pair-fed equivalent to WT mice (pair-fed ob/ob). Mice were maintained at 32 °C (thermoneutral) from 6 to 18 weeks of age to minimize differences in resting energy expenditure. ob/ob mice were heavier, had more abdominal white adipose tissue (WAT), and were hyperglycemic compared with WT mice. Femur length, bone mineral content (BMC) and bone mineral density, and midshaft femur cortical thickness were lower in ob/ob mice than in WT mice. Cancellous bone volume (BV) fraction was higher but indices of bone formation and resorption were lower in ob/ob mice compared with WT mice; reduced bone resorption in ob/ob mice resulted in pathological retention of calcified cartilage. Pair-fed ob/ob mice were lighter and had lower WAT, uterine weight, and serum glucose than ob/ob mice. Similarly, femoral length, BMC, and cortical thickness were lower in pair-fed ob/ob mice compared with ob/ob mice, as were indices of cancellous bone formation and resorption. In contrast, bone marrow adiposity, calcified cartilage, and cancellous BV fraction were higher at one or more cancellous sites in pair-fed ob/ob mice compared with ob/ob mice. These findings indicate that the skeletal abnormalities caused by leptin deficiency are markedly attenuated in morbidly obese ob/ob mice.

Introduction

Leptin, a polypeptide hormone secreted by adipocytes, is best known for its role in the regulation of appetite and energy metabolism (Mistry et al. 1997, Myers 2004). Rodents deficient in leptin signaling, due to either an inability to generate leptin (ob/ob mice) or the signaling form of the leptin receptor (db/db mice and fa/fa rats), become morbidly obese (Clement 2000). Their excess weight is the result of a combination of hyperphagia and reduced thermogenesis (Hwa et al. 1996). In addition to morbid obesity, leptin deficiency is associated with hypogonadism (Barkan et al. 2005), elevated corticosteroid levels (Saito & Bray 1983), and impaired thermoregulation (Trayhurn & James 1978).

Leptin-deficient mice and leptin-receptor-deficient mice and rats have reduced overall bone mass, reduced longitudinal bone growth (Steppan et al. 2000, Kishida et al. 2005), and decreased bone formation (Gat-Yablonski & Phillip 2008). In addition, impaired bone resorption in ob/ob and db/db mice results in mild osteopetrosis (Turner et al. 2013), a condition that may contribute to the reported reductions in bone quality observed in these animals (Ealey et al. 2006, Kimura et al. 2012). Impaired bone resorption may also account for the age- and bone-dependent variation in cancellous bone volume (BV) observed in leptin-signaling-deficient mice; the mutant mice have been reported to have normal to increased cancellous BV fraction in lumbar vertebra and decreased to increased cancellous BV fraction in long bones (Hamrick et al. 2004, Iwaniec et al. 2007, Williams et al. 2011). The skeletal abnormalities observed in mice and rats with defective leptin signaling indicate that leptin plays a role in normal skeletal growth, maturation, and turnover (Steppan et al. 2000, Kishida et al. 2005, Iwaniec et al. 2007, Bartell et al. 2011, Turner et al. 2013). However, it is not clear to what extent the skeletal phenotype of ob/ob mice is affected by additional factors associated with morbid obesity.

Although rare in humans, leptin signaling deficiency occurs as a result of loss-of-function mutations in the genes for leptin and the leptin receptor (Friedman & Halaas 1998). More common causes for low levels of leptin signaling in humans include anorexia and starvation (Muller et al. 2009, Dardeno et al. 2010). At the other end of the spectrum, diet-induced leptin resistance, associated with chronically elevated leptin levels, mimics many of the metabolic consequences of leptin deficiency on energy metabolism and is thought to contribute to the development of morbid obesity (Jung & Kim 2013). Thus, changes in leptin levels and/or leptin sensitivity could have physiological effects on bone metabolism.

In order to better understand the specific actions of leptin in regulating bone mass, density, and architecture, we performed a study in which differences in body weight gain between rapidly growing WT and ob/ob mice were prevented by housing all mice at thermoneutral temperature (32 °C) to minimize differences in resting energy expenditure (Trayhurn 1979) and pair-feeding the mutant mice to the level for WT mice to equalize food intake.

Materials and methods

Experimental design

The experimental protocols were approved by the Institutional Animal Care and Use Committee in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Four-week-old female ob/ob (n=20) and homozygous WT (+/+) littermate (n=11) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). At 6 weeks of age, all animals were transferred from normal room temperature (22 °C) to thermoneutral temperature (32 °C) and maintained at thermoneutrality on a 12 h light:12 h darkness cycle for the duration of the study (12 weeks). Feed (Teklad 8604, Harlen Laboratories, Indianapolis, IN, USA) and water were provided to WT mice (n=11) and a group of ob/ob mice (n=10) that were allowed to eat and drink ad libitum. A second group of ob/ob mice (n=10) were pair-fed to the WT mice; the ob/ob mice were fed an amount of food equivalent to the group mean for WT mice.

Feed consumption was measured daily in WT mice and weekly in ob/ob mice allowed to feed ad libitum. Body weight was recorded weekly for all mice. The fluorochrome declomycin (20 mg/kg, Sigma) was administered at initiation of the study and the fluorochrome calcein (20 mg/kg, Sigma) was administered 4 days and 1 day before killing to label the mineralizing bone. All mice were fasted overnight. Mice were then anesthetized with 2–3% isoflurane delivered in oxygen, bled by cardiac puncture, and glucose measured using a glucometer (Life Scan, Inc., Milpitas, CA, USA). Serum was collected and stored at −80 °C for measurement of global markers of bone turnover. Uteri and abdominal white adipose tissue (WAT) were excised and weighed. Femora and lumbar vertebrae were removed and stored in 70% ethanol for analysis using dual-energy X-ray absorptiometry (DXA), microcomputed tomography (μCT), and histomorphometry. Tibiae were removed, cleaned of soft tissue, frozen in liquid nitrogen, and stored at −80 °C for mRNA isolation and gene expression analysis.

Serum chemistry

Serum osteocalcin was measured using a mouse Gla-osteocalcin High Sensitive EIA kit obtained from Clontech. Serum CTx was measured using a mouse CTx ELISA kit obtained from Life Sciences Advanced Technologies.

Dual-energy X-ray absorptiometry

Femoral bone mineral content (BMC, mg), area (cm2), and bone mineral density (BMD, g/cm2) were determined ex vivo using DXA (Piximus, Lunar Corp., Madison, WI, USA).

Microcomputed tomography

Nondestructive three-dimensional evaluation of bone microarchitecture was carried out using μCT (Bouxsein et al. 2010). Femora and lumbar vertebrae were scanned in 70% ethanol using a Scanco μCT40 scanner (Scanco Medical AG, Basserdorf, Switzerland) at a voxel size of 12×12×12 μm (55 kVp X-ray voltage, 145 μA intensity, and 200 ms integration time). Evaluations were carried out with the filtering parameters sigma and support set at 0.8 and 1 respectively. Bone segmentation was carried out at a threshold of 245 (scale, 0–1000) determined empirically. Total femur was evaluated followed by the evaluation of cortical bone in the midshaft femur and cancellous bone in the distal femur metaphysis and epiphysis. Automated contouring was used to delineate cortical bone from non-bone. Then, all cortical slices were examined visually for potential inclusion of cancellous struts originating from the endocortex (extremely rare at this site) and were manually removed when present. For cortical bone, 20 slices (0.24 mm) of bone were evaluated, and cross-sectional tissue volume (TV) (cortical and marrow volume, mm3), cortical volume (mm3), marrow volume (mm3), and cortical thickness (μm) were measured. The polar moment of inertia was determined as a surrogate measure of bone strength in torsion. Architectural parameters are expressed using the standard three-dimensional nomenclature. For the femoral metaphysis, 40 slices (0.48 mm) of cancellous bone were evaluated. The entire cancellous compartment was assessed in the femoral epiphysis. Analysis of lumbar vertebra included the entire region of cancellous bone between the cranial and caudal growth plates. Manual contouring was used to delineate cancellous from cortical bone in the femur metaphysis, femur epiphysis, and vertebral body. Cancellous bone measurements in femur and lumbar vertebra included BV fraction (BV/TV, %), connectivity density (per mm3), trabecular number (per mm), trabecular thickness (μm), and trabecular separation (μm) (Thomsen et al. 2005).

Histomorphometry

The histological methods used here have been described in detail previously (Iwaniec et al. 2008). In brief, the distal femur was dehydrated in a graded series of ethanol and xylene and embedded undecalcified in modified methyl methacrylate. Sections (4 μm thick) were cut with a vertical bed microtome (Leica/Jung 2165) and affixed to slides precoated with a 1% gelatin solution. Sections were mounted unstained for the measurement of fluorochrome labels. For cell-based measurements, sections were stained for tartrate-resistant acid phosphatase and counterstained with toluidine blue (Sigma). All data were collected using the OsteoMeasure System (OsteoMetrics, Inc., Atlanta, GA, USA). The sampling site for the distal femoral metaphysis was located 0.25–1.25 mm proximal to the growth plate. The entire cancellous compartment was evaluated in the distal femoral epiphysis and body of lumbar vertebra.

Fluorochrome-based measurements of bone formation included the following parameters: i) mineralizing perimeter (mineralizing perimeter/bone perimeter: cancellous bone perimeter covered with double plus half single label normalized to bone perimeter, %), ii) mineral apposition rate (the distance between two fluorochrome markers that comprise a double label divided by the 3 day interlabel interval, μm/day), and iii) bone formation rate (bone formation rate/bone perimeter: calculated by multiplying mineralizing perimeter by mineral apposition rate normalized to bone perimeter, μm2/μm per year).

Declomycin label retained in the regions of interest (mm label/mm2 tissue area) in the distal femur epiphysis and lumbar vertebra was measured as a dynamic index of bone resorption in ob/ob and pair-fed ob/ob mice. This method is based on the principle that declomycin label was incorporated into bone at equivalent rates in both groups of ob/ob mice when the fluorochrome was administered before treatment initiation and differences measured at study termination reflect the effect of treatment on resorption of the fluorochrome-labeled bone. This method has been described previously (Westerlind et al. 1997). We did not measure declomycin label in WT mice because almost no label was retained in this group at the termination of the study.

Cell-based measurements included osteoblast perimeter, osteoclast perimeter, adipocyte number, and adipocyte area. Osteoblast perimeter was determined as a percentage of total bone perimeter lined by a palisade of plump cuboidal cells located immediately adjacent to the thin layer of osteoid in direct physical contact with the bone perimeter (osteoblast perimeter/bone perimeter, %). Osteoclast perimeter was determined as the percentage of cancellous bone perimeter covered by multinucleated (two or more nuclei) cells with the acid phosphatase-positive (red-stained) cytoplasm (osteoclast perimeter/bone perimeter, %). Cartilage area in the distal femur metaphysis and epiphysis was measured and expressed as a percentage of the cancellous bone area. The intense metachromatic (purple) staining of proteoglycans and glycosaminoglycans with toluidine blue was used to identify the cartilage within trabeculae. Adipocyte number and area were also measured and expressed as bone marrow adiposity (tissue area occupied by adipocytes: adipocyte area/tissue area, %), and adipocyte density (number of adipocytes/mm2). Adipocytes were identified morphologically as large (>75 μm2) circular or oval-shaped cells bordered by a prominent cell membrane lacking cytoplasmic staining due to alcohol extraction of intracellular lipids during processing. This method has been previously validated by fat extraction and analysis (Menagh et al. 2010). All histomorphometric data are expressed using the standard two-dimensional nomenclature (Dempster et al. 2013) to distinguish two-dimensional histomorphometric measurements from three-dimensional μCT measurements.

Gene expression

Tibiae were pulverized with a mortar and pestle in liquid nitrogen and then further homogenized in Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's protocol, and mRNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). The expression of 84 genes related to osteogenic differentiation was determined using the Mouse Osteogenesis RT2 Profiler PCR Array (Qiagen) according to the manufacturer's protocol. Gene expression was normalized to Gapdh and relative quantification was determined by the ΔΔCt method using RT2 Profiler PCR Array Data Analysis software version 3.5 (Qiagen).

Statistical analysis

Mean responses for individual variables were compared among the WT, ob/ob, and pair-fed ob/ob groups using separate one-way ANOVA, with Tukey's procedure for pairwise multiple comparisons. The required conditions for the valid use of ANOVA models were assessed using Levene's test for homogeneity of variance, plots of residuals versus fitted values, normal quantile plots, and the Anderson–Darling test of normality. A modified F test was used when the assumption of equal variance was violated, with Welch's two-sample t-test used for pairwise comparisons (Welch 1951). The Kruskal–Wallis nonparametric test was used when only the normality assumption was violated, in which case the Wilcoxon–Mann–Whitney test was used for pairwise comparisons. Longitudinal data on body weight were analyzed using a random intercept linear mixed model that allowed distinct linear rates of change for the WT and pair-fed ob/ob groups and a quadratic time trend for the ob/ob group. The Hommel method for maintaining the familywise error rate at 5% was used to adjust for multiple comparisons (Hommel 1988). Data analysis was performed using R version 2.12. Gene expression analysis was performed using RT2 Profiler PCR Array Data Analysis version 3.5 (Qiagen). Gene expression is reported as mean fold change. All other data are expressed as mean±s.e.m.

Results

The effects of pair-feeding (to match food intake in WT mice) on body weight and cumulative food intake, and on abdominal WAT weight, blood glucose levels, uterine weight, and serum osteocalcin and CTx levels at killing in leptin-deficient ob/ob mice housed at thermoneutral temperature are shown in Fig. 1. Six-week-old ob/ob mice were heavier than their age-matched WT littermates at the start of treatment and gained more weight than WT mice during the 12-week study period (Fig. 1A and B). ob/ob mice were hyperphagic consuming, on average, twice as much feed as WT mice (Fig. 1C). At termination of study, the ob/ob mice had more abdominal WAT (Fig. 1D) and were hyperglycemic (Fig. 1E) and hypogonadal (Fig. 1F) compared with WT mice.

Figure 1
Figure 1

Effects of morbid obesity on body weight (A), cumulative body weight change (B), cumulative food intake (C), abdominal white adipose tissue (WAT) weight (D), blood glucose (E), uterine weight (F), and serum osteocalcin (G) and CTx levels (H) in leptin-deficient ob/ob mice. Values are expressed as mean±s.e.m., n=10–11/group. ND, no data due to insufficient serum for measurement of CTx in pair-fed ob/ob mice. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Pair-fed ob/ob mice had a lower body weight and body weight gain, lower cumulative food intake, lower abdominal WAT weight, lower blood glucose levels, and slightly lower uterine weight than ob/ob mice. Pair-feeding abolished differences in weight gain and blood glucose levels between WT and ob/ob mice. However, the pair-fed ob/ob mice were still heavier, had more WAT, and were hypogonadal compared with WT mice.

Effects of pair-feeding on serum markers of bone turnover in ob/ob mice

Serum levels of osteocalcin (Fig. 1G) and CTx (Fig. 1H) were lower in ob/ob mice than in WT mice. Pair-fed ob/ob mice also had lower serum osteocalcin levels than either WT or ob/ob mice. Insufficient serum was available for the measurement of CTx in pair-fed ob/ob mice.

Effects of pair-feeding on bone mass and architecture in ob/ob mice

The effects of pair-feeding on femur BMC, area and BMD, and on femur length, BV, and cortical and cancellous bone architecture in ob/ob mice are given in Table 1. ob/ob mice had lower femur BMC, bone area, and BMD than WT mice. Pair-fed ob/ob mice had lower BMC and bone area than WT or ob/ob mice. Significant differences in BMD were not detected between ob/ob and pair-fed ob/ob mice.

Table 1

Effects of morbid obesity on femoral bone mineral content and density, femoral cortical and cancellous bone microarchitecture, and vertebral cancellous bone microarchitecture in leptin-deficient ob/ob mice

End pointWTob/obob/ob (pair-fed)ANOVA Pa
Dual-energy X-ray absorptiometry
 Total femur
 BMC (g)0.023±0.0000.019±0.000b0.017±0.000b,c0.0001
 Bone area (cm2)0.43±0.010.40±0.00b0.37±0.01b,c0.0001
 BMD (g/cm2)0.053±0.0010.048±0.000b0.047±0.000b0.0003
Microcomputed tomography
 Total femur
 Length (mm)14.9±0.114.2±0.1b13.4±0.1b,c<0.0001
 Bone volume (mm3)18.5±0.316.8±0.3b15.0±0.3b,c<0.0001
 Midshaft femur (cortical bone)
 Cross-sectional volume (mm3)0.41±0.010.40±0.010.42±0.010.4220
 Cortical volume (mm3)0.20±0.000.18±0.00b0.17±0.00b,c0.0008
 Marrow volume (mm3)0.22±0.000.23±0.000.25±0.01b,c0.0012
 Cortical thickness (μm)195±3176±3b159±2b,c0.0001
 Polar moment of inertia (mm4)0.36±0.010.33±0.010.33±0.010.4220
 Distal femur metaphysis (cancellous bone)
 Bone volume/tissue volume (%)11.2±0.813.2±0.5b18.1±0.5b,c0.0004
 Connectivity density (1/mm3)106±9173±7b190±8b<0.0001
 Trabecular thickness (μm)44±141±145±0c0.0365
 Trabecular number (per mm)4.8±0.15.2±0.1b5.7±0.1b,c0.0001
 Trabecular separation (μm)210±4191±4b173±4b,c0.0013
 Distal femur epiphysis (cancellous bone)
 Bone volume/tissue volume (%)31.2±0.636.1±0.6b36.6±0.9b0.0013
 Connectivity density (1/mm3)155±4254±8b261±7b<0.0001
 Trabecular thickness (μm)64±160±1b58±1b0.0001
 Trabecular number (per mm)5.5±0.16.7±0.1b6.7±0.1b0.0004
 Trabecular separation (μm)179±2138±3b138±3b<0.0001
 Lumbar vertebra (cancellous bone)
 Bone volume/tissue volume (%)21.8±1.031.5±0.8b34.2±0.5b<0.0001
 Connectivity density (1/mm3)198±10364±15b191±5c<0.0001
 Trabecular thickness (μm)49±150±0.554±0.4b,c0.0010
 Trabecular number (per mm)4.6±0.16.0±0.1b5.8±0.1b0.0004
 Trabecular separation (μm)215±5160±3b163±3b0.0005

The Hommel method for maintaining the familywise error rate at 5% was used to adjust for multiple comparisons.

Different from WT, P<0.05.

Different from ob/ob, P<0.05.

Femur length and BV

ob/ob mice had shorter femurs and a lower femoral BV than WT mice. Pair-fed ob/ob mice had a lower femur length and a lower total femur BV than WT or ob/ob mice.

Cortical bone in midshaft femur

ob/ob mice had lower cortical BV and cortical thickness than WT mice. Pair-fed ob/ob mice had lower midshaft femur cortical BV and cortical thickness, and greater marrow volume than WT or ob/ob mice. Significant differences in cross-sectional volume and polar moment of inertia were not detected among groups.

Cancellous bone in distal femur metaphysis

ob/ob mice had higher cancellous BV fraction, connectivity density, and trabecular number, and lower trabecular separation than WT mice. Differences in trabecular thickness were not detected between the two groups. Pair-fed ob/ob mice had higher cancellous BV fraction and trabecular number, and lower trabecular separation than WT mice or ob/ob mice, and a higher connectivity density than WT mice. Trabecular thickness in pair-fed ob/ob mice was higher than that in ob/ob mice but did not differ from that in WT mice.

Cancellous bone in distal femur epiphysis

ob/ob mice and pair-fed ob/ob mice had higher BV fraction, connectivity density, and trabecular number, and lower trabecular thickness and separation than WT mice. Differences between ob/ob mice and pair-fed ob/ob mice were not detected for any epiphyseal endpoint measured.

Cancellous bone in lumbar vertebra

The effects of pair-feeding on cancellous bone mass and architecture in lumbar vertebra in ob/ob mice are given in Table 1. ob/ob mice had higher cancellous BV fraction, connectivity density, and trabecular number, and lower trabecular separation than WT mice. Pair-fed ob/ob mice had a higher BV fraction than WT mice and a tendency (P<0.1) towards a higher BV fraction than ob/ob mice. Pair-fed ob/ob mice also had higher trabecular thickness than WT or ob/ob mice. Connectivity density was lower in pair-fed ob/ob mice than in ob/ob mice but did not differ from that for WT mice. Trabecular number was higher and trabecular separation was lower in pair-fed ob/ob mice than WT mice. Differences in trabecular number and separation were not detected between ob/ob mice and pair-fed ob/ob mice.

Effects of pair-feeding on bone histomorphometry in ob/ob mice

Cancellous bone in distal femur metaphysis

The effects of pair-feeding on dynamic and static cancellous bone histomorphometry and marrow adiposity in the distal femur metaphysis in ob/ob mice are shown in Fig. 2. ob/ob mice had lower mineralizing perimeter (Fig. 2A), mineral apposition rate (Fig. 2B), bone formation rate (Fig. 2C), and osteoblast perimeter (Fig. 2D) compared with WT mice. Pair-fed ob/ob mice had lower mineralizing perimeter, mineral apposition rate, bone formation rate, and osteoblast perimeter than WT or ob/ob mice. Differences in osteoclast perimeter (Fig. 2E) were not detected with treatment. However, cartilage area (Fig. 2F), an index of impaired bone resorption, was higher in both groups of ob/ob mice compared with WT mice. Bone marrow adiposity (Fig. 2G) tended (P<0.1) to be greater in ob/ob mice than WT mice and was greater in pair-fed ob/ob mice than in WT or ob/ob mice. Adipocyte density (Fig. 2H) was higher in pair-fed ob/ob mice than in ob/ob mice. Representative photomicrographs of marrow adipocytes in the three groups of mice are shown in Fig. 3A, B and C.

Figure 2
Figure 2

Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in distal femur metaphysis in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), cartilage area (F, cartilage area/tissue area, Cg.Ar/T.Ar), marrow adiposity (G, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (H, number of adipocytes/tissue area, N.Ad/T.Ar). Values are expressed as mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. a*Different from WT mice, P<0.1. bDifferent from ob/ob mice, P<0.05.

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Figure 3
Figure 3

Representative photomicrographs of i) marrow adiposity in distal femur metaphysis in WT mouse (A), ob/ob mouse (B), and pair-fed ob/ob mouse (C), ii) cartilage remnants in distal femur epiphysis in WT mouse (D), ob/ob mouse (E), and pair-fed ob/ob mouse (F), and iii) declomycin label administered at study initiation in distal femur epiphysis in WT mouse (G), ob/ob mouse (H), and pair-fed ob/ob mouse (I).

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Cancellous bone in distal femur epiphysis

The effects of pair-feeding on dynamic and static cancellous bone histomorphometry and marrow adiposity in the distal femur epiphysis in ob/ob mice are shown in Fig. 4. ob/ob mice had lower mineralizing perimeter (Fig. 4A), bone formation rate (Fig. 4C), and osteoblast perimeter (Fig. 4D) than WT mice. Pair-fed ob/ob mice had lower mineralizing perimeter, bone formation rate, and osteoblast perimeter than WT or ob/ob mice. Osteoclast perimeter (Fig. 4E) was higher in ob/ob mice and pair-fed ob/ob mice than in WT mice. Retention of declomycin label (Fig. 4F), an index of decreased bone resorption, was higher in pair-fed ob/ob mice than in ob/ob mice. ob/ob mice had greater cartilage area (Fig. 4G), marrow adiposity (Fig. 4H), and adipocyte density (Fig. 4I) than WT mice. Pair-fed ob/ob mice had higher cartilage area, marrow adiposity, and adipocyte density than WT or ob/ob mice. Representative photomicrographs showing cartilage and declomycin labelling in the three groups are shown in Fig. 3D, E, F and G, H, I respectively.

Figure 4
Figure 4

Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in distal femur epiphysis in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), declomycin label perimeter (F, declomycin label perimeter/tissue area, declomycin L.Pm/T.Ar), cartilage area (G, cartilage area/tissue area, Cg.Ar/T.Ar), marrow adiposity (H, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (I, number of adipocytes/tissue area, N.Ad/T.Ar). Values are mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Cancellous bone in lumbar vertebra

The effects of pair-feeding on dynamic and static cancellous bone histomorphometry and on bone marrow adiposity in lumbar vertebra are shown in Fig. 5. ob/ob mice had lower mineralizing perimeter (Fig. 5A), bone formation rate (Fig. 5C), and osteoblast perimeter (Fig. 5D) than WT mice. Pair-fed ob/ob mice had lower mineralizing perimeter, mineral apposition rate (Fig. 5B), bone formation rate, and osteoblast perimeter than WT or ob/ob mice. Significant differences in osteoclast perimeter (Fig. 5E) were not detected among the three groups. However, retention of declomycin label (Fig. 5F) was higher in pair-fed ob/ob mice than in ob/ob mice. ob/ob mice had greater marrow adiposity (Fig. 5G) and adipocyte density (Fig. 5H) than WT mice. Pair-fed ob/ob mice had higher marrow adiposity and adipocyte density than WT or ob/ob mice.

Figure 5
Figure 5

Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in lumbar vertebra in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), declomycin label perimeter (F, declomycin label perimeter/tissue area, declomycin L.Pm/T.Ar), marrow adiposity (G, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (H, number of adipocytes/tissue area, N.Ad/T.Ar). Values are expressed as mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Effects of pair-feeding on gene expression in tibia

The effects of pair-feeding on expression of 84 genes related to osteogenesis in tibia are shown in Fig. 6. Compared with WT mice, expression levels of 24 genes were altered in ob/ob mice (seven higher and 17 lower) and 26 genes in pair-fed ob/ob mice (ten higher and 16 lower). The expression levels of 14 genes were altered in common in ob/ob and pair-fed ob/ob mice. Compared with ob/ob mice, expression levels of 11 genes (three higher and eight lower) were altered in pair-fed ob/ob mice. ob/ob mice exhibited reductions in several key genes related to osteoblast and osteoclast differentiation and function, including Sp7/osterix (−2.5-fold), NfkB1 (−1.2-fold), alkaline phosphatase (Alpl) (−1.5-fold), Cd11b (Itgam) (−1.3-fold), and osteocalcin (Bglap) (−1.8-fold). ob/ob mice also exhibited reductions in genes related to cartilage formation and maturation, including col2a1 (−1.5-fold) and col10a1 (−1.7-fold). Genes whose expressions were elevated include Bmpr1a (+1.3-fold) and Smad4 (+1.3-fold). Compared with WT mice, pair-fed ob/ob mice expressed even lower levels of osteocalcin (−3.8-fold) than those observed in ob/ob mice and, in addition, had lower mRNA levels for the osteoclast differentiation factor Csf1 (−1.5-fold) and osteoblast differentiation factor Runx2 (−1.4-fold). Furthermore, compared with WT mice, pair-fed ob/ob mice had higher gene expression levels for the cytokines/growth factors Tnfa (TNF) (+1.6-fold) and Tgfb2 (+1.3-fold).

Figure 6
Figure 6

Effects of morbid obesity on expression of osteogenesis-related genes in tibia. The expressions of a panel of 84 osteogenesis-related genes in the tibia of WT mice (n=6), ob/ob mice (n=5), and ob/ob mice pair-fed to the WT mice (n=5) were determined using a mouse osteogenesis gene expression array. Left panel shows hierarchical clustering analysis of genes with significantly different gene expression. The magnitude of gene expression was represented by green and red bars, indicating decreased and increased expression respectively. The normalized mean fold change of ob/ob mice and pair-fed ob/ob mice relative to the WT mice was shown in the right panel. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

Citation: Journal of Endocrinology 223, 1; 10.1530/JOE-14-0224

Discussion

We investigated the contribution of excessive weight gain to the skeletal phenotype of leptin-deficient ob/ob mice. Mice were housed at 32 °C and a group of ob/ob mice was pair-fed to WT mice. Housing mice at 32 °C, which is within the thermoneutral range for WT and ob/ob mice, was expected to minimize the differences in the resting energy expenditure and pair-feeding ob/ob mice with respect to WT mice was expected to equalize energy intake (Knehans & Romsos 1982, Rafael & Herling 2000). The parallel increase in body weight gain in WT mice and ob/ob mice pair-fed with respect to WT mice over the 12-week duration of study demonstrates the efficacy of this approach for preventing the development of morbid obesity in leptin-deficient mice.

Consistent with results from earlier studies (Hamrick et al. 2004, Ealey et al. 2006, Iwaniec et al. 2007, Williams et al. 2011), ob/ob mice exhibited a mosaic skeletal phenotype associated with decreased bone formation due to reduced osteoblast number and activity, and defective osteoclast function. Femurs from ob/ob mice were smaller and had thinner cortices than those from WT mice. Furthermore, serum osteocalcin levels, mRNA levels of osteocalcin and Sp7 (osterix), osteoblast perimeter, and dynamic indices of bone formation were lower in ob/ob mice than in WT mice. However, cancellous BV fraction in both distal femur and lumbar vertebra was higher in the ob/ob mice. The higher cancellous BV fraction was associated with i) greatly reduced serum CTx levels, ii) reductions in mRNA levels of Nfkb1, Itgam, and Icam1, iii) retention of calcified cartilage, and iv) retention of fluorochrome label administered at the start of the study, all indicative of a defect in bone resorption. Osteoclast perimeter was either unaffected (distal femur metaphysis and lumbar vertebra) or increased (distal femur epiphysis), indicating that reduced bone resorption in ob/ob mice was not due to an inability to generate osteoclasts. Taken together, these findings further support our previous observation that ob/ob mice exhibit mild osteopetrosis. Osteopetrosis due to a defect in osteoclast function may be responsible for the poor bone quality (Ealey et al. 2006) and defective tooth eruption (Batt 1978, 1992) reported in ob/ob mice.

Pair-feeding accentuated many of the abnormalities in bone size, mass, microarchitecture, gene expression, and turnover typically observed in ob/ob mice. Thus, chronic excessive weight gain typically present in ob/ob mice reduces the magnitude of skeletal effects associated with leptin deficiency. We did not adjust μCT-derived cortical bone data for body weight differences because the association between body weight and bone size and density in growing mice varies with bone and bone compartment and this association is altered by leptin (Iwaniec et al. 2009). However, it is clear that the weight gain due to hyperphagia in ob/ob mice was associated with increases in cortical bone accrual but that these gains were not commensurate with the magnitude of body weight gain.

The mechanisms for the partial protective effect of morbid obesity on bone in ob/ob mice are likely to be multifactorial. We have previously reported a strong positive correlation between body weight and total femur bone mass in ob/ob mice (Iwaniec et al. 2009). This finding provides strong evidence that the higher total and cortical bone mass in mice allowed to feed ad libitum compared with pair-fed ob/ob mice are, at least in part, attributable to higher body weight. Although there have been significant recent advances in our understanding of the regulation of bone metabolism by mechanotransduction, the molecular mechanism for this positive effect of weight on bone mass is largely unknown. In addition to direct effects of increased mechanical loading, changes in levels of hormones and/or growth factors may mediate additional effects of increased weight on the skeleton (Robling 2012).

We did not measure the levels of insulin, glucocorticoids, or norepinephrine, factors known to be dysregulated in ob/ob mice (Knehans & Romsos 1982, Dubuc et al. 1985, Kim & Romsos 1990, Rafael & Herling 2000) and known to influence bone metabolism. However, housing food-restricted ob/ob mice at thermoneutral temperature has been reported to reduce abnormalities in blood glucose, insulin, glucocorticoids, and norepinephrine turnover. In contrast, ob/ob mice allowed to feed ad libitum have been reported to remain hyperglycemic and hyperinsulinemic (Lindstrom 2007). We confirmed that blood glucose levels in pair-fed ob/ob mice did not differ from those of WT mice and were much lower than those of ob/ob mice allowed to feed ad libitum. The contribution of excess insulin secretion and other factors associated with morbid obesity to the protective effects of excess body weight (Martineau-Doize et al. 1986, Ituarte et al. 1988, Pun et al. 1989, Hickman & McElduff 1990, Yaturu 2009) on cortical bone microarchitecture in ob/ob mice requires further investigation.

Leptin is required for gonadotropin-releasing hormone secretion (Donato et al. 2011). As a consequence, ob/ob mice are severely hypogonadal (Chehab et al. 2002). The resulting deficiency in sex steroids would be expected to play a role in the pathological skeletal changes associated with leptin deficiency because sex steroids, especially estrogen, contribute to the sexual dimorphism of the female skeleton and regulate bone turnover balance in adults (Turner et al. 1989, 1994, Rickard et al. 2008). Gonadal dysfunction typically results in elevated bone turnover and cancellous osteopenia. The difference between the skeletal responses to gonadal hormone insufficiency resulting from gonadectomy (high turnover) and from leptin-deficiency (low turnover) is perplexing. However, estrogen has a tonic effect on bone resorption and leptin is required for normal osteoclast function. As a consequence, the high bone turnover generally associated with estrogen deficiency may be attenuated in ob/ob mice due to impaired osteoclast function. We speculate that the requirement for leptin in normal osteoclast function may serve as a counter-regulatory mechanism to prevent excessive bone loss during fasting.

We did not measure sex steroid levels but the higher uterine weight in ob/ob mice fed ad libitum compared with pair-fed ob/ob mice implies that morbid obesity results in a small increase in circulating estrogen. Higher estrogen levels in blood or adipose tissue may contribute to the protective skeletal effects of morbid obesity (Turner et al. 1994, Turner 1999, Gennari et al. 2011). Aromatase, the enzyme responsible for conversion of androgens to estrogens, is expressed by adipocytes (Gennari et al. 2011). Thus, adipose-derived estrogen is a plausible factor contributing to the skeletal differences between pair-fed mice and ob/ob mice allowed to feed ad libitum.

Initially heavier ob/ob mice pair-fed with respect to WT mice remained heavier than WT mice during the entire study period. We chose to equalize energy availability rather than body weight in this study, because bone accrual in rapidly growing rodents is strongly associated with energy availability and bone loss accompanies weight loss in adults (Devlin et al. 2010, Turner & Iwaniec 2011). Low leptin levels induced by caloric restriction are associated with diminished bone mass accrual, whereas partial leptin signaling deficiency induced in mice allowed to feed ad libitum by treatment with a leptin antagonist increased body weight and bone mass accrual (Solomon et al. 2014). However, administration of leptin increases bone formation in adult ob/ob mice in spite of weight loss (Bartell et al. 2011, Turner et al. 2013). Thus, both adequate energy availability and leptin play important roles in bone accrual in growing mice.

In contrast to peripheral fat depots, which act as dynamic energy reservoirs to maintain circulating triglyceride and free fatty acid levels during feeding and fasting, marrow fat increases with severe weight loss as well as weight gain (Bredella et al. 2011, Devlin 2011). It is therefore unlikely that the bone marrow fat depot serves as an important energy reservoir for peripheral tissues. There is, however, evidence that bone marrow adipocytes function as negative regulators of hematopoietic lineage cell differentiation (Gimble et al. 1996, Dorshkind et al. 2009, Naveiras et al. 2009). Bone marrow fat is increased in ob/ob mice and this increase is reversed by leptin treatment (Hamrick et al. 2004, 2005, Bartell et al. 2011). Leptin stimulates hematopoiesis (Fantuzzi & Faggioni 2000, Trottier et al. 2012), an action that may result from inhibition of bone marrow adipogenesis, increased lipolysis, and direct stimulatory actions on hematopoietic cells. Adipocytes and osteoblasts are derived from the same bone marrow mesenchymal precursor and a reciprocal association between the osteoblast number and the adipocyte number has often been noticed, giving rise to the hypothesis that either bone marrow adipocytes may antagonize osteoblast production or, alternatively, generation of adipocytes may preclude generation of osteoblasts (Akune et al. 2004). Results from other studies indicate that the adipocyte and osteoblast number in bone marrow can be independently regulated (Menagh et al. 2010, Iwaniec & Turner 2013). Bartell et al. (2011) reported that i.c.v. administration of leptin to ob/ob mice increases expression of Sp7, a transcription factor critical for osteoblast differentiation. Our findings of low expression of SP7 in ob/ob mice support the hypothesis that leptin acts to regulate bone formation by controlling mesenchymal cell decision.

In this study, pair-feeding with respect to WT mice resulted in a further increase in bone marrow adiposity in leptin-deficient ob/ob mice. Of interest is our finding that pair-fed ob/ob mice had elevated mRNA levels of Cd36; CD36 (fatty acid translocase) promotes adipocyte differentiation and adipogenesis (Christiaens et al. 2012). The observed increase in CD36 may play a role in the increased bone marrow adiposity observed in the pair-fed ob/ob mice. Additional factors regulating bone marrow adiposity that may be influenced by leptin levels and obesity include growth hormone and insulin-like growth factor 1 (IGF1). In rats, treatment with parathyroid hormone (whose actions on bone are largely mediated by IGF1) prevented the increase in bone marrow adiposity associated with severe energy restriction (Turner & Iwaniec 2011), whereas growth hormone treatment dramatically reduced excessive bone marrow adiposity in severely hypoleptinemic hypophysectomized rats (Menagh et al. 2010). In both of these studies, treatment had no effect on serum leptin levels. ob/ob mice, however, have decreased circulating growth hormone and impaired growth hormone signaling (Luque et al. 2007), probably contributing to retention of fat within the bone marrow. High circulating insulin levels in morbidly obese ob/ob mice may further contribute to the suppression of growth hormone synthesis and release (Luque & Kineman 2006). However, IGF1 is produced and secreted by adipocytes (Kloting et al. 2008) and higher levels of IGF1 are detected in obese subjects (Frystyk et al. 1999). Low leptin levels resulting from severe energy restriction are associated with increased growth hormone secretion, decreased circulating levels of IGF1, and end-organ resistance to growth hormone (Grinspoon et al. 1995, Thissen et al. 1999, Douyon & Schteingart 2002, Munoz & Argente 2002, Inagaki et al. 2008). Thus, disturbances in growth hormone, IGF1, and insulin signaling as well as leptin deficiency may contribute to the increase in bone marrow adiposity in ob/ob mice. Chronic hyperphagia and excessive weight gain typically observed in ob/ob mice may partially compensate for leptin deficiency and reduced bone marrow adiposity by altering growth hormone and insulin levels and sensitivity.

In summary, leptin deficiency resulted in decreased femur length, BMC, BMD, and cortical BV and thickness. In contrast, the cancellous BV fraction was increased in distal femur and lumbar vertebra. The profound changes in bone mass, density, and microarchitecture were associated with reduced linear bone growth, increased bone marrow adiposity, altered expression of osteogenic genes, decreased osteoblast number, and decreased osteoclast function. Prevention of morbid obesity in ob/ob mice further exacerbated many of the abnormalities associated with leptin deficiency. As morbid obesity normally present in ob/ob mice attenuates the abnormal skeletal phenotype associated with leptin deficiency, the role of leptin in regulating postnatal bone growth, maturation, and turnover may have been underestimated.

Declaration of interest

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

Funding

This work was supported by the National Institutes of Health (NIH) (AR 060913), the National Aeronautics and Space Administration (NASA) (NNX12AL24), and the United States Department of Agriculture (USDA) (38420-17804).

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  • WilliamsGACallonKEWatsonMCostaJLDingYDickinsonMWangYNaotDReidIRCornishJ2011Skeletal phenotype of the leptin receptor-deficient db/db mouse. Journal of Bone and Mineral Research2616981709. (doi:10.1002/jbmr.367)

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  • YaturuS2009Diabetes and skeletal health. Journal of Diabetes1246254. (doi:10.1111/j.1753-0407.2009.00049.x)

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    Effects of morbid obesity on body weight (A), cumulative body weight change (B), cumulative food intake (C), abdominal white adipose tissue (WAT) weight (D), blood glucose (E), uterine weight (F), and serum osteocalcin (G) and CTx levels (H) in leptin-deficient ob/ob mice. Values are expressed as mean±s.e.m., n=10–11/group. ND, no data due to insufficient serum for measurement of CTx in pair-fed ob/ob mice. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

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    Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in distal femur metaphysis in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), cartilage area (F, cartilage area/tissue area, Cg.Ar/T.Ar), marrow adiposity (G, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (H, number of adipocytes/tissue area, N.Ad/T.Ar). Values are expressed as mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. a*Different from WT mice, P<0.1. bDifferent from ob/ob mice, P<0.05.

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    Representative photomicrographs of i) marrow adiposity in distal femur metaphysis in WT mouse (A), ob/ob mouse (B), and pair-fed ob/ob mouse (C), ii) cartilage remnants in distal femur epiphysis in WT mouse (D), ob/ob mouse (E), and pair-fed ob/ob mouse (F), and iii) declomycin label administered at study initiation in distal femur epiphysis in WT mouse (G), ob/ob mouse (H), and pair-fed ob/ob mouse (I).

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    Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in distal femur epiphysis in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), declomycin label perimeter (F, declomycin label perimeter/tissue area, declomycin L.Pm/T.Ar), cartilage area (G, cartilage area/tissue area, Cg.Ar/T.Ar), marrow adiposity (H, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (I, number of adipocytes/tissue area, N.Ad/T.Ar). Values are mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

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    Effects of morbid obesity on cancellous bone histomorphometry and marrow adiposity in lumbar vertebra in leptin-deficient ob/ob mice. Shown are mineralizing perimeter (A, mineralizing perimeter/bone perimeter, M.Pm/B.Pm), mineral apposition rate (B, MAR), bone formation rate (C, bone formation rate/bone perimeter, BFR/B.Pm), osteoblast perimeter (D, osteoblast perimeter/bone perimeter, Ob.Pm/B.Pm), osteoclast perimeter (E, osteoclast perimeter/bone perimeter, Oc.Pm/B.Pm), declomycin label perimeter (F, declomycin label perimeter/tissue area, declomycin L.Pm/T.Ar), marrow adiposity (G, adipocyte area/tissue area, Ad.Ar/T.Ar), and adipocyte density (H, number of adipocytes/tissue area, N.Ad/T.Ar). Values are expressed as mean±s.e.m., n=10–11/group. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

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    Effects of morbid obesity on expression of osteogenesis-related genes in tibia. The expressions of a panel of 84 osteogenesis-related genes in the tibia of WT mice (n=6), ob/ob mice (n=5), and ob/ob mice pair-fed to the WT mice (n=5) were determined using a mouse osteogenesis gene expression array. Left panel shows hierarchical clustering analysis of genes with significantly different gene expression. The magnitude of gene expression was represented by green and red bars, indicating decreased and increased expression respectively. The normalized mean fold change of ob/ob mice and pair-fed ob/ob mice relative to the WT mice was shown in the right panel. aDifferent from WT mice, P<0.05. bDifferent from ob/ob mice, P<0.05.

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    • Export Citation
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    • Export Citation
  • YaturuS2009Diabetes and skeletal health. Journal of Diabetes1246254. (doi:10.1111/j.1753-0407.2009.00049.x)