Bone abnormalities in adolescent leptin-deficient mice

Regulatory Peptides 136 (2006) 9 – 13 www.elsevier.com/locate/regpep

Bone abnormalities in adolescent leptin-deficient mice Kafi N. Ealey, Debbie Fonseca, Michael C. Archer, Wendy E. Ward ⁎ Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College Street, Toronto, Ontario, Canada M5S 3E2 Received 23 August 2005; received in revised form 24 March 2006; accepted 28 April 2006 Available online 9 June 2006

Abstract Ob/ob and db/db mice have different aberrations in leptin signaling that both lead to abnormalities in bone mineral density (BMD), and bone histological and histomorphometric outcomes. A few studies have directly compared bone metabolism in ob/ob and db/db mice, and biomechanical strength properties that are surrogate measures of fracture risk, have not been extensively studied. This study compared bone mineral content (BMC), BMD and biomechanical strength properties of femurs and lumbar vertebrae among 10 week old male ob/ob, db/db and C57Bl/6 wildtype (WT) mice. Femurs and lumbar vertebrae were specifically studied to determine if trabecular and cortical bone are regulated by leptin in a similar manner in ob/ob and db/db mice. Femurs of ob/ob and db/db mice had lower BMC, BMD and biomechanical strength properties, including peak load, compared to WT mice. In contrast, lumbar vertebrae BMC and BMD did not differ among genotypes, nor did the peak load from compression testing of an individual lumbar vertebra differ among groups. These findings suggest that leptin deficiency in adolescent male mice first results in changes in femurs, a representative long bone, and alterations in lumbar vertebrae may occur later in life. © 2006 Elsevier B.V. All rights reserved. Keywords: Leptin; Bone; Mice; Bone mineral density; Biomechanical bone strength

1. Introduction Leptin, the product of the ob gene, is a 16 KDa protein secreted by adipocytes that is involved in appetite control and food intake and energy regulation [1]. Leptin exerts its main effects in regulating energy balance in the hypothalamus, by binding to its receptor (ObR), encoded by the db gene [2,3]. Two mouse models of obesity are known to result from spontaneous mutations in the leptin pathway. Ob/ob mice have a mutation in the leptin gene resulting in functional inactivation of the protein and undetectable circulating leptin levels [1]. Db/db mice have a mutation that affects the long form transcript of the leptin receptor (ObRb) resulting in no expression of ObRb, and highly elevated levels of circulating leptin [3,4]. Leptin is thought to be primarily an antiobesity hormone although, it has also been shown to play a role in lipid metabolism [5], cardiovascular and renal function [2], liver function, alcohol metabolism [6] and bone metabolism [7]. Studies examining the role of leptin in regulating bone mass have reported contradictory findings. Leptin-deficient ob/ob mice are reported to have lower femur bone density compared to lean ⁎ Corresponding author. Tel.: +1 416 946 7366; fax: +1 416 978 5882. E-mail address: [email protected] (W.E. Ward). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.04.013

wildtype controls [8,9]. Similarly, leptin receptor-deficient Zucker rats have decreased bone mass in comparison to controls [10,11] and osteopenia has been reported in db/db mice [12]. In a recent study comparing the bone mineral content (BMC) and bone mineral density (BMD) in ob/ob mice and lean controls, it was reported that 6 month old ob/ob mice had decreased femur BMD, BMC and quadriceps mass but increased BMC and BMD in the lumbar spine, suggesting that the effects of leptin on cortical and trabecular bone differ [13]. These findings have been confirmed by other studies reporting that the decreased bone mass observed in leptin-deficient animals is corrected when animals are treated with leptin, suggesting that leptin itself has an anabolic effect on bone [8,14]. Ducy et al. [15], however, reported that leptin deficiency is associated with increased femur and vertebrae BMD in both ob/ob and db/db female mice [15], while there were no differences in peak load between female ob/ob mice and lean controls. Furthermore, intracerebroventricular infusion of leptin resulted in significant reductions in bone mass in these mice [15]. Others have also reported that leptin decreases bone mass and that this effect is mediated centrally [7,16]. Evidence for a role of leptin in bone metabolism from in vitro studies is also conflicting. Some studies suggest that leptin increases the proliferation and mineralization of osteoblasts and

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inhibits apoptosis of osteoblasts [17,18]. Furthermore, both human and rat osteoblasts have been reported to express functional ObR isoforms [17,19], suggesting that leptin may have local effects on bone. Others, however, have been unable to detect the presence of the leptin receptor in mouse primary osteoblasts [15] and report that osteoblasts do not respond to leptin treatment, thus supporting a centrally mediated effect of leptin. The objective of this study was to compare BMC, BMD and functional measures of bone strength such as biomechanical strength properties of femurs and lumbar vertebrae in ob/ob and db/db mice. We chose to study adolescent male mice since others have reported the effects of leptin on bone in older male animals or in females and it is possible that this could explain some of these discrepancies. Two distinct sites of the skeleton, femurs and lumbar vertebrae, were specifically studied to determine if bones differing in their ratio of trabecular and cortical bone are regulated by leptin in a similar manner in these animals.

which permanent damage will occur in the bone and resilience is a measure of the amount of energy that the femur absorbs until the yield point is reached. Stiffness is a measure of the extrinsic rigidity of the femur. Peak load is the maximal force the femur can withstand before fracturing and toughness is a measure of the energy absorbed until the peak load is reached. Yield load, resilience and stiffness are predominately the measures of the contribution of mineral to bone strength whereas peak load and toughness are predominately the measures of the contribution of matrix to bone strength. Femur length was measured using digital calipers (Cedarlane Laboratories Ltd., Hornby ON) and from this measurement, the midpoint of the femur was determined. The posterior sides of the femur were placed on two base supports of a bending jig separated by 6 mm, with the midpoint directly under the crosshead. The crosshead was lowered at a constant speed of 2 mm/min until fracture occurred. To minimize shear forces during the fracture test, the tips of the bending jig are rounded.

2. Materials and methods

2.3.3. Compression testing of lumbar vertebra 4 (LV4) Compression testing of an individual lumbar vertebra (LV4) was performed to determine differences in a skeletal site that contains predominately trabecular bone. As previously described [20], LV4 was isolated from the rest of the vertebra, soft tissue was excised and spinous processes removed, and subsequently each individual LV4 was placed in the center of a smooth stainless steel plate. A compression force was applied to the vertebra by lowering a second suspended stainless steel plate at a constant rate of 2 mm/min. Visual inspection during each test verified that a vertebra remained in a stable position. Compression force was applied and the peak load of compression was determined to be the first peak on the load–deformation curve.

2.1. Animals Thirty male mice (C57Bl/6–ob/ob, C57Bl/6–db/db and C57Bl/6 lean wildtype mice, n = 10/group) (Jackson Laboratories, Bar Harbor, Maine) were killed at 10 weeks of age and femurs and lumbar vertebrae 1–4 (LV1–LV4) were collected for the measurement of BMC, BMD and biomechanical strength properties. All animal procedures were approved by the University of Toronto Animal Ethics Committee. 2.2. Femur and lumbar vertebrae 1–4 (LV1–LV4), bone mineral content (BMC) and bone mineral density (BMD)

2.4. Statistical analyses At necropsy, femurs and intact lumbar vertebrae (LV1–LV4) were excised and stored at −70 °C until analyses were performed. As previously described [20], individual whole femurs and intact vertebra (LV1–LV4) were dissected free of all soft tissue, placed on a plastic tray and scanned in air using PIXImus dual energy X-ray absorptiometry (GE Medical Systems, Mississauga, Ontario, Canada). 2.3. Biomechanical strength testing 2.3.1. Femurs and lumbar vertebra 4 (LV4) Biomechanical strength properties of right femurs and LV4 were determined using a material testing system (Model 4442 Universal Testing System; Instron Corp., Canton, MA) and a specialized software program (Instron Series IX Automated Materials Tester—Version 8.15.00; Instron Corp). Bones were rehydrated in physiological saline (9 g NaCl/L) for 4 h at room temperature prior to testing. 2.3.2. Three-point bending at the femur midpoint To determine the biomechanical strength properties at a site rich in cortical bone, a three-point bending test was performed on femurs [20]. The following properties that contribute to the strength of the femur were determined: yield load, resilience, stiffness, peak load, and toughness. Yield load is the point in

All data are expressed as means ± SEM. Statistical analyses were performed using one-way ANOVA with Student–Newman Keul's test used for comparison of multiple means. Differences were considered significant if p b 0.05. To determine the contribution of BMC to the various biomechanical strength properties, linear regression analyses were performed with BMC as the independent variable and the strength properties as dependent variables in separate analyses. All statistical analyses were performed using SigmaStat (Jandel Corp., San Rafael, CA, USA). 3. Results 3.1. Body weights Body weights were significantly higher (p b 0.05) among ob/ ob and db/db mice compared to WT mice at necropsy (ob/ ob = 50 ± 1 g; db/db = 45 ± 1 g; WT = 24 ± 1 g). 3.2. BMC, BMD and biomechanical strength properties 3.2.1. Whole femur BMC and BMD Whole femur BMC and BMD of ob/ob and db/db mice were significantly lower than WT mice (Table 1). The biomechanical

K.N. Ealey et al. / Regulatory Peptides 136 (2006) 9–13 Table 1 Whole femur BMC and BMD, and biomechanical strength properties at the femur midpoint WT Whole femur BMC (mg) BMD (mg/cm2) Femur midpoint Yield load (N) Resilience (J × 10− 4) Peak load (N) Toughness (J × 10− 3) Ultimate stiffness (N/mm)

ob/ob

db/db

25 ± 1a 63 ± 1a

20 ± 1b 56 ± 1b

19 ± 1b 55 ± 1b

8.9 ± 0.6a 3.6 ± 0.6a 17.1 ± 0.5a 11.6 ± 0.8a 123.7 ± 4.8a

5.7 ± 0.2b 2.2 ± 0.1b 12.6 ± 0.3b 8.0 ± 1.0b 83.3 ± 3.9b

6.8 ± 0.7b 2.5 ± 0.3ab 13.7 ± 0.4b 6.6 ± 0.6b 84.4 ± 5.8b

Values are expressed as mean ± SEM. Values with different letters within a row are significantly different, p b 0.05.

strength properties of femurs such as yield load, peak load, toughness and stiffness were also significantly lower (p b 0.05) among ob/ob and db/db mice compared to WT mice (Table 1). Resilience was lower (p b 0.05) among ob/ob and db/db mice compared to WT mice, but values for db/db mice did not quite reach statistical significance (Table 1). 3.2.2. BMC and BMD of lumbar vertebrae 1–4 (LV1–LV4); and peak load of lumbar vertebra 4 (LV4) There was no difference in BMC and BMD of LV1–LV4 among WT, ob/ob and db/db mice (Table 2). Similarly, the peak load of LV4 did not differ among the three groups (Table 2). 3.2.3. Contribution of whole femur BMC to each of the femur biomechanical strength properties Whole femur BMC was a significant (p b 0.05) contributor to femur biomechanical strength properties including yield load, resilience, peak load, toughness and stiffness (Fig. 1). 4. Discussion The main objective of this study was to determine whether leptin affects functional measures of bone strength in adolescent mice at two distinct sites of the skeleton, the femur and lumbar vertebrae. Novel findings from this study include the profound negative effect of leptin deficiency in both ob/ob and db/db mice on a number of biomechanical strength measures of femurs that are surrogate measures of fracture risk. Other studies have reported alterations in femur BMC and/or BMD and histology [7– 9,13,15] but none have specifically focused on biomechanical bone strength properties of femurs and lumbar vertebrae of ob/ob and db/db mice. The data suggest that both the mineral and matrix component in femurs were compromised in ob/ob and db/db mice based on the BMC, BMD and biomechanical strength data. Of the biomechanical strength properties measured, yield load and stiffness are indicators of the contribution of mineral to bone strength whereas peak load is an indicator of the contribution of bone matrix proteins to bone strength. The fact that all of these outcomes were lower among ob/ob and db/db mice suggests that both the deposition of mineral and matrix is hindered in femurs of these growing mice. Moreover, the significant correlation among

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femur BMC and each of the femur biomechanical strength properties measured provides further evidence that both phases of bone, matrix and mineral, were correspondingly compromised. Femurs that contain a higher proportion of cortical bone than lumbar vertebrae, were adversely affected by leptin deficiency. Lumbar vertebrae, however, were resistant to leptin deficiency since BMC and BMD of intact lumbar vertebrae 1–4 (LV1–LV4) did not differ among groups. Of functional relevance is the finding that an individual vertebra (LV4) had a similar peak load, indicative of resistance to compression fracture, regardless of genotype. Our findings in this study differ from that of Hamrick et al. [13] in which BMC and BMD of lumbar vertebrae (LV2–LV3) were increased in male obese mice. It is possible that these discrepant findings are due to a difference in the age of the animals, 10 weeks in our study versus 6 months in Hamrick et al. [13]. Thus, differences in lumbar vertebrae may only be manifested later in life after peak bone mass is achieved at approximately 4 months of age [21]. Ducy et al. [15] reported higher BMD at femur and lumbar vertebrae at 3 and 6 months of age in female mice. These findings suggest that gender differences are likely to be important when comparing findings among studies. Several studies that report femur outcomes in male ob/ob mice of a similar age to those in the present study do not report lumbar vertebrae outcomes [8,9]. The precise mechanisms by which leptin deficiency induces these abnormalities in bone metabolism require further investigation. It is likely that the ObRa receptor is not directly involved with the effects of leptin on bone since db/db mice express this receptor and yet experienced similar abnormalities in bone metabolism as ob/ob mice that have no circulating leptin. There is evidence that the lower femur BMD in ob/ob male mice, is at least in part, due to higher rates of bone resorption mediated by higher levels of serum 1,25-dihydroxyvitamin D3 resulting from the elevated activity of 1α-hydroxylase and 24-hydroxylase [9]. Interestingly, Steppan et al. [8] demonstrated that the administration of leptin to male ob/ob mice resulted in an increase in both trabecular and cortical bone mass of femurs. In conclusion, both ob/ob and db/db mice, despite markedly greater body weights than WT mice, have lower femur but not lumbar vertebrae BMC and BMD. The fact that the ratio of cortical and trabecular bone varies dramatically between femur and lumbar vertebrae suggests that these types of bone are affected differently by a state of leptin deficiency at an early stage of development (10 weeks of age) but this requires confirmation. Moreover, the finding that peak load, the maximum force a bone can withstand before fracture, is lower at the femur midpoint, provides evidence of the functional consequences of leptin deficiency. With the increasing prevalence of obesity, particularly among children, the findings from the present study using growing ob/ob or db/db mice may prove useful for understanding the role and Table 2 BMC and BMD of LV1–LV4 and peak load of LV4

BMC (mg) BMD (mg/cm2) Peak load (N)

WT

ob/ob

db/db

28 ± 1 56 ± 1 33.1 ± 2.2

31 ± 1 58 ± 2 38.3 ± 2.3

29 ± 2 56 ± 2 37.0 ± 2.7

Values are expressed as mean ± SEM.

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Fig. 1. a–e. Correlation between femur BMC. a. yield load; b. resilience; c. peak load; d. toughness; and e. stiffness. Symbols used: ο WT; □ ob/ob; Δ db/db.

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