No Adaptations in Bone of Leptin- Deficient ob/ob Mice in Response to Loading

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Introduction

Leptin is a peptide hormone produced predominantly by whi- te fat cells [1,47]. The mature protein, encoded by the obesity (ob) gene localized in human and mouse 7 and 6 chromosomes, respectively, is a 16 kDa non-glycosylated protein. Leptin levels in the blood are proportional to adipose tissue mass. In- itially discovered as a central regulator of appetite and energy expenditure, leptin levels can be considered as a signal to the body regarding its energy reserves. Although elevated leptin levels are present in obesity, it is likely that the physiological importance of leptin is that low levels indicate a state of star- vation [2].

Recent data suggest that leptin may regulate a variety of other physiological processes, such as insulin action [7], hemopoie- sis [5], immune function [28], reproduction [40], angiogenesis [26], and bone development and remodeling [19].

However, it is not clear whether leptin is a stimulator or an inhibitor of bone growth in humans. Some investigators noted

a positive relationship between serum leptin levels and bone mineral density (BMD). Iwamoto et al. described a weakly correlation of serum leptin level with bone mineral density of pelvis and left leg in the premenopausal women and Odebasi et al. found that there was no correlation between plasma leptin concentrations and BMD values in healthy postmenopausal woman but a weak correlation was observed in postmenopausal woman with osteoporosis [23,35]. Whereas others observed a negative relationship. Blum et al. suggest that for a given body weight, a higher proportion of fat and a higher serum leptin concentration have negative associations with bone mass in premenopausal women [6]. Sato et al. suggest that an increase in serum leptin reduces bone formation and de- creases BMD in adult men [42].

Furthermore, no associations between serum leptin levels and BMD have been reported, which further confounds the inter- pretation of leptin’s effect on bone mass. Martini et al. conclude that serum leptin have no direct effect on bone mass and bone turnover in healthy postmenopausal women [32] and Rauch et al. appear that leptin has less influence on the mature than on the growing skeleton [41].

Only a few studies in humans have examined the direct effect of leptin administration on BMD. Ogueh et al. [36] noted a Autor:Hansjoerg

Heep¹, Christian Wede- meyer¹, Jie Xu², Sebas- tian Hofmeister¹, Marius von Knoch¹

Schlagworte:Leptin, trabekulärer und korti- kaler Knochen, MikroCT, biomechanische Belas- tung

Keywords:leptin, cortical und trabecular bone, microCT, biomechanical loading

Zitierweise dieses Bei- trages: BIOmaterialien 2008; 9 (1/2): S. 18-25

Leptin was found to be involved in regulation of bone development and remodeling. This study was performed to evaluate the effects of biomechanical loading due to body weight in leptin-defient ob/ob mice at the same pubertal stage. No significant difference was found between the two groups for all the parameters (p>0.05) suggesting that the presence of leptin may be an important pre-condition for positive correlation between loading and bone mass. Furthermore, concordance of bone formation was found among appendicular re- gions, but was not found between the axial and appendicular re- gions.

No Adaptations in Bone of Leptin- Deficient ob/ob Mice in Response to Loading

Keine Anpassung des Knochens von Leptin-defizienten ob/ob-Mäusen bei biomechanischer Belastung

1 Department of Orthopaedics, University of Duisburg-Essen, Pattbergstrasse 1-3, 45239 Essen, Germany

2 Department of Orthopaedics, The second affiliated hospital of Sun Yat-Sen University Guangzhou, China

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significant negative correlation between fetal blood levels of leptin and cross-linked carboxyl-terminal telopeptide of Ty- pe 1 collagen (a marker of bone resorption). A modest negative correlation was also noted between leptin levels and the concentration of carboxyl-terminal pro-peptide of Type 1 collagen (a marker of bone formation), but the researchers spe- culated that the overall effect of leptin on fetal bone metabolism was to increase bone mass by decreasing bone resorption [36]. One study reported an increase in bone mass and decre- ase in bodyweight after long-term leptin therapy in an obese 9-year-old girl with congenital leptin deficiency [12]. That might show the positive effect on bone formation in early development. Simha et al. concluded that the effect of leptin on bone metabolism may depend on the stage of life in humans because there were no effects of leptin on osteoblastic or oste- oclastic activity [43]. Larger clinical studies were therefore necessary to clarify this hypothesis. In few recent reports the animal model was a useful surrogate in investigating the mechanism of leptin. Both leptin and its receptors were found in murine fetal cartilage and bone template, as well as in the growth plate [27]. In addition, leptin increased both proliferation and differentiation of the chondrocyte population of skeletal growth centers in organ cultures. The results of Ma- or et al. indicate that leptin acts as a skeletal growth factor with a direct peripheral effect on skeletal growth centers and they speculate that the high circulating levels of leptin in obese children might contribute to their growth [31]. Nakajima et al. show that the growth plate chondrocytes possess leptin receptors (Ob-Rs), and leptin enhance chondrocyte proliferation and subsequent cell differentiation [34].

All these strongly supported the theory that leptin could sti- mulate bone growth in the early stages of life.

Weight-bearing exercise has been shown to have an advanta- geous effect on BMD. Chen et al. demonstrate results of the histomorphometric analysis after 9 weeks of exercise. They showed that the periosteal labeled surface, mineral apposition rate, and bone formation rate were profoundly increased in aged female rats [9]. Iwamoto et al. findings suggested that in the mature osteopenic rat, there was a beneficial effect of mo- derate running exercise with adequate calcium intake on bone mass only in a weight-bearing long bone, the tibia. The mechanism for increased bone mass appeared to be both decreased bone resorption and increased bone formation in cancellous bone and increased bone formation in cortical bone [24]. Yeh et al. found that in the aged rat, by 9 weeks, exercise increases BMD in the tibia and in the vertebrae [46].

The phenomena of bone loss with disuse [45] and bone accru- al with in vivo loading [17] certainly emphasizes the importance of mechanical loading for bone formation and mainte- nance in normal subjects without the knock-out ob gene. Me- chanical loading of bones via ground reaction forces and muscle pulling on the bones are generally accepted as necessary stimuli to attain peak BMD and maintain bone homeos- tasis. In general, mechanical loading stress on bones causes tissue deformation within the bone (due to strain) that stimulates the bone to adapt by remodeling to accommodate these demands, ultimately improving resistance to osteoporosis.

While previous studies have shown that bone parameters improved with weight-bearing exercise in normal subjects, we were interested in the co-influence of biomechanical loading on body weight and bone metabolism in subjects with leptin deficiency. The aim of this study was to investigate the differen- tial effects of different degrees of weight-bearing on trabecular and cortical bone formation in the mice without the stimulation of leptin. We chose the obese gene in mice model because this gene is known to effect both bone bone mass and body mass.

We hypothesized that biomechanical loading due to body weight might be an ignored factor which has lead to the contradictory results regarding BMD in studies with mice which were not known to be deficient in leptin or had inactive leptin receptors.

Materials and Methods

Animals

C57BL/6J-Lep^ob(ob/ob) female mice with an autosomal re- cessive mutation on chromosome 6 were received from the Janvier Laboratory (Le Genest St Isle, France) at five weeks of age. The animal experiment was appoved by the Univer- sity`s ethic committee and the local authorities according to the official guidelines. The animals were housed one per ca- ge with access to water ad libitum and a standard rodent diet (8640 Harlan Teklad 22/5[W]; Harlan Teklad, Madison, WI, USA) containing 1.13% calcium and 0.94% phosphorus.

The animals were maintained under conditions of a twelve- hour light and dark cycle with the light switched on at 6.00 a.m.. Food intake and body weight were recorded daily. The- re were two experimental groups divided according to the provision of food. Group A included 20 ob/ob mice with access to food ad libitum, the other 20 ob/ob mice in Group B received a limited amount of food (6 gr. each day). All the animals were euthanized by mechanical procedure at the age of twenty weeks.

Micro-CT

A high resolution micro-CT (SkyScan 1072, Aartselaar, Bel- gium) was used to perform qualitative and quantitative analysis of the limb and the vertebral body. All samples were scanned by a commercially available microcomputed tomographic scanner at the Department of Cardiology of the West German He- art Centre at the University of Duisburg-Essen.

Technical specifications of the Micro-CT

The micro-CT system is based on a scanner developed for high- resolution imaging (up to 4 µm cubic voxels) of sample sizes up to 2 cm³. The scanner uses a field x-ray tube with an 8 µm spot-seize and expected lifetime of > 10,000 hours. The tube is operated at between 20 and 100 kiloelectron volts and a current of up to 100 micro amperes. For scanning, the samples are placed between the microfocus X-ray source and a Char- ge-Coupled-Device (CCD) detector (matrix size: 1024x1024 pi- xels, field of view: 25 mm²). In order to prevent samples from moving during scanning, the imbs and vertebral bodies are placed in a tightly fitting rigid plastic tube. In the scanner’s chamber the specimens are placed on a stack of computer- controlled precision stages which are rotated in equiangular steps of 0.9° around an angle of 180°. When the object is placed between the x-ray source and the CCD-detector, the cone- beam of X-rays passes the object and then hits the CCD-detector producing 2D-X-ray images. A personal computer is used to control the scanner and store the CCD image data recorded at each angle of view during the scanning process.

Tomographic Image Reconstruction

The X-ray projection data of the scanned samples are then submitted to the resident reconstruction program (Cone-beam Reconstruction, Skyscan, Aartselaar, Belgium), which is based on a Feldkamp filtered back projection algorithm [48] resulting in a volume image of up to 10,243 voxels, each cubic voxel being 4–19 µm on one side, depending on how much of the specimen had to be imaged.

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Cancellous bone assessment by micro-CT

Three regions of interest (ROIs) from the vertebrae, proximal femur and tibia were selected for structural analysis of the cancellous bone. The ROI for the vertebrae was 0.5×0.5×0.5mm³ in the middle of Lumbar 3. A cubic region of 0.5×0.5×0.5mm³ in the metaphysis of the tibia was adjacent to (1mm away from) the growth plate and femoral head. Bone volume ratio (BV/TV) was calculated by adding the number of voxels re- presenting mineralized trabecular bone divided by the total volume, and expressed as a percentage. The bone surface ratio (BS/BV) was calculated by adding the surface components of the mineralized bone phase directly from the 3-D data and dividing by the total bone phase volume. Trabecular thickness (Tb. Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were also based directly on the 3-D data using medial axis transformation and distance transformation.

Cortical bone assessment by micro-CT

In the femur, a cubic region of 2.5×2.5×2.5mm³in the middle of the shaft adjacent to the femoral crest was selected. This region, which could include the whole cycle of the cortical bone, contained no trabecular bone. Bone volume (BV) and bone surface (BS) were calculated by adding the number of voxels and the surface components of the mineralized bone from the 3-D data respectively. Cortical thickness (C.Th) was calculated by the formula:

C.TH = 2x ,

introduced by Bagi CM et al [3], which can be explained by:

area of a ring = thickness of ring × length of middle line =

thickness × .

Determining the thickness via 3D parameters allowed a more robust and precise determination than calculating them directly on individual slices only.

Statistical analysis

Data were analyzed and assessed using SPSS software (ver- sion 12.0; SPSS Institute Inc, Chicago). Descriptive statistics of all variables were determined including the mean and standard deviation of each group. The difference of all parameters between the two groups was assessed by the Student`s t-Test because all parameters (were normal distributed which was tested with the Kolmogorov Smirnov test). Pearson’s correlation coefficient was used to assess the relationship between all the trabecular bone parameters of femur, vertebra and tibia.

A value of p≤0.05 was considered to be statistically significant.

Results

No death or health deterioration occurred during this study.

The body weight at each time point in the two groups is shown in Figure 1.

Animals with an ad libitum diet (Group A) were found to increase body weight significantly at the age of six weeks in com- parison with the lean mice (Group B). From this time point on the difference constantly increased. At the age of twenty weeks the obese mice were almost twice as heavy as the lean mice.

Micro-CT measurements were obtained for assessment of the morphological changes in the two groups (Fig. 2). Examination of trabecular bone in the limbs and spine revealed a statistically insignificant difference between the two groups. High weight- bearing insignificantly improved all trabecular bone parameters in the obese mice (Table 1).

Weight-bearing showed no correlation with the cortical bone parameters, including BV, BS and thickness of femoral shaft.

One-way ANOVA did not reveal any statistically significant difference between the parameters in the two groups (Table 1).

Figure 3 provided scatter plots of each of the morphological pa- Fig. 1: The curves show the change in body-weight in the two groups. In Group A the animals had an ad libitum diet, the animals in Group B a controlled diet. Inter-group difference was already significant at the age of six weeks (p<0.05).

Fig. 2. Three-dimensional images of all the ROIs of the mice in Groups A and B at the age of 20 weeks. The ROI for the vertebrae was 0.5x0.5x0.5mm³ in the middle of Lumbar 3. A cubic region of 0.5x0.5x0.5mm³in the metaphysis of the tibia was adjacent to (1mm away from) the growth plate and femoral head.

BV BS

outercircumference + innercircumference 2

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rameters of the trabecular bone in the limbs and vertebrae.

The related regression results are summarized in Table 3. All the femoral parameters exhibited linear relations with those of the tibia, but non-linear relations with the vertebra.

Discussion

A great deal of research has confirmed that increased biomechanical loading due to increased body weight contributes to the increased bone dimensions and mass observed in not only our animal model but also obese humans. Frost et al. des- cribe that an increased loading of long bones produces the greatest mechanical stresses on the subperiosteal surface and stimulates bone formation by subperiosteal expansion [14].

For example, loading in the playing arm in racquet sports in- duces significant increases in bone dimensions and mass [18].

The effect of weight gain on bone loading has not only been examined in adults but also in postmenopausal women. Beck et al. show a significant increase in hip cortical section modulus due to periosteal expansion [4]. A qCT (quantitative compu- tertomography) study in healthy children suggested that weight-bearing and mechanical stresses are important deter- minants of cortical bone mass, whereas trabecular bone density is influenced by hormonal factors associated with sexual development [33].

A study of bone biomechanics in adult rats with diet-induced obesity showed significantly greater bone strength in the obese rats than in the controls. The cross-sectional geometry and ultimate fracture load of the femur were higher in the obese rats than in the controls [8].

In our study, however, there were no significant differences in trabecular and cortical bone mass between the leptin-deficient obese mice and the leptin-deficient control mice. In other words, it seemed that increased biomechanical loading due

to increased body weight did not contribute to increased bone dimensions and mass in the leptin-deficient subjects.

We considered that our results did not negate the conclusion of the other research mentioned above regarding the positive effect of biomechanical loading on bone mass, as these studies did not focus on leptin-deficient subjects. Numerous types of obesity were studied: one of them was congenital leptin deficiency, which is described in the human obesity gene map [38]. Oral et al. reported on a group of nine patients whose obesity was attributed to low serum leptin levels, and whose condition improved after leptin replacement [37]. Another study investigated subjects with a higher serum leptin level. Much research has concentrated on the positive effect of leptin in not only decreasing food intake but also increasing bone formation by modulating the sympathetic nervous system [10].

During the same phase, the increased biomechanical loading due to increased body weight contributed to the increased bone mass as a co-influence. Hla et al. and the Early Postmeno- pausal Intervention Cohort (EPIC) Study Group show that the increased bone mineral content (BMC) in childhood obesity was revealed both in the weight-bearing limbs and the unlo- aded arms [22]. The new finding of our study, which focused on the co-influence of biomechanical loading in subjects with leptin-deficiency, is that it confirmed the presence of leptin as the pre-condition for a positive correlation between loading and bone mass.

We could, of course, not eliminate the possible effect of other factors on bone mass, such as estrogen. Estrogen was confirmed to be present in postmenopausal obese females at a rela- tively higher serum level because adipocytes were a major si- te of estrogen production in these subjects. Heshmati et al. demonstrate that endogenous residual estrogen levels determine bone resorption even in the late postmenopausal women [21].

Position Group BV/TV (%)

Mean S.D.

BS/BV (1/mm) Mean S.D.

Tb.Th (mm) Mean S.D.

Tb.Sp (mm) Mean S.D.

Tb.N(1/mm) Mean S.D.

Femur Group A

(diet ad libitum) Group B (control diet)

P-value

60.66 6.13

62.69 6.53

0.317

36.10 3.81

36.70 3.92

0.837

0.094 0.0069

0.093 0.0075

0.905

0.098 0.012

0.092 0.011

0.088

6.42 0.45

6.74 0.55

0.051

Tibia Group A

P-value

53.31 4.16

54.64 7.48

0.494

37.89 2.65

38.56 3.74

0.520

0.094 0.0063

0.093 0.0084

0.505

0.13 0.015

0.13 0.022

0.526

5.67 0.36

5.90 0.52

0.118 Vertebrae Group A

P-value

31.79 6.06

34.97 8.17

0.171

45.46 4.09

43.90 4.92

0.283

0.085 0.0067

0.085 0.0062

0.925

0.17 0.034

0.16 0.028

0.358

4.09 0.78

3.75 0.59

0.134 Table 1: Cross-section structural geometric properties of the femur, tibia and vertebrae were evaluated using micro-CT. Note: Summary of morphome- tric characteristics in the two groups which were different in body weight-bearing. No statistically significant difference was detected between the two groups (p>0.05).

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Khosla et al. suggest that in contrast to traditional belief, age- related bone loss may be the result of estrogen deficiency not just in postmenopausal women, but also in men [25]. But the mice in our study were no more than twenty weeks old (in the pubertal stage) and without ovariotomy. Thus, estrogen as a confounding factor in our study is not very likely. There is so far no agreement regarding the positive effect of leptin in the early stages of life. The data gathered during some research studies appear conflicting or even contradictory. Ducy et al.

observed that three- and six- month old ob/ob mice, which were deficient in leptin, and db/db mice, which have a mutated and inactive leptin receptor, had higher trabecular bone mass associated with a higher mineral apposition rate when compared with their wild-type litter-mates [11]. On the other hand, Foldes et al. showed that obese fa/fa rats, which also had a mutated leptin receptor, had a lower bone mass with shorter and lighter femurs when compared with their normal litter-mates [13]. Lower femoral BMD and osteocalcin (a marker of bone formation) serum were described in fa/fa rats, as well as decreased trabecular bone volume, trabecular thickness and trabecular number, as measured both by micro-CT and histomorphometry. Picherit et al. have compared femoral bone density and biochemical markers of bone metabolism in male and female fatty (leptin-resistant) Zucker rats and their lean homozygous controls at 3 and 6 months of age [39]. Tamasi et al. used female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls at 9 and 15 weeks of age. They suggest that leptin exerts a positive effect on bone, based on the observed skeletal phenotype of the Zucker (fa/fa) rat [44]. The reasons for these discrepancies remain unclear.

In our study, concordance of bone formation was found among appendicular regions, but not between the axial and appendicular regions. Hamrick et al [20] found that ob/ob mice show increased bone content, density and trabecular bone volume in the lumbar spine which was consistent with the results of Du- cy et al [11]. Due to the limitation of the design this study, we did not assess the change from each time point of growth.

Even so, the discrepancy of bone formation between limb and spine indicated the different effect of leptin on bone formation in axis and appendicular regions, or the bone formation of the spine took place earlier than that of the limb and was stable at the time of our assessment. Large clinical studies are necessary to consolidate leptin’s role in the physiology of human bone.

Abstract

Problemstellung: Das Hormon Leptin reguliert die Reifung und Entwicklung des Knochens. Jedoch nicht bekannt ist der Ein- fluß von Leptin auf das wachsende Skelett. Unsere Studie untersucht die Auswirkung der biomechanischen Belastung des Körpergewichtes von Leptin-defizienten ob/ob-Mäusen in der Pubertät.

Methodik: Beginnend mit einem Alter von 5 Wochen wurden zwei Gruppen von je 20 weiblichen ob/ob-Mäusen untersucht.

Mit einem Alter von 20 Wochen war das Körpergewicht der Gruppe A, die keine Einschränkung des Essverhaltens erfuhren, signifikant schwerer als in der Gruppe B unter kontrollierter Diät (p<0.05). Die trabekulären und kortikalen Strukturen des Knochens wurden mittels MikroCT untersucht.

Ergebnisse: Unter Berücksichtigung, daß Leptin einen wichti- Fig. 3: Plots of the correlation between the femoral, tibial and vertebral regions, respectively for trabecular parameters. The correlation between the femoral and tibial regions was represented by an open triangle (LL), between the femoral and vertebral regions by a filled square (IZ.

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gen Faktor für eine positive Korrelation zwischen der Belastung und der Knochenmasse darstellt, zeigt sich keine signifikante Unterscheidung der beiden Gruppen in allen Parametern (p>0.05). Jedoch zeigen sich Übereinstimmungen der knö- chernen Strukturen entlang der Extremitäten, die sich nicht im Vergleich zwischen den Extremitäten und des Achsenske- letts nachweisen lassen.

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Group BV(mm³)

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Thickness(mm) Mean S.D.

Group A

(diet ad libitum) 0.28 0.025 5.274 0.300 0.108 0.005

Group B

(control diet) 0.277 0.015 5.553 1.608 0.103 0.014

P-value 0.176 0.337 0.120

Table 2: Cross-sectional structural geometric properties in the femoral shaft were evaluated using micro-CT.

Parameter r P-value Equation

Femur vs tibia BV/TV (%) BS/BV (1/mm)

Tb.Th (mm) Tb.Sp (mm)

0.525 0.569 0.512 0.320

<0.05

y= 31.857 + 0.552 x y= 20.801 + 0.479 x y= 0.0437 + 0.529 x y= 0.0772 + 0.521 x Femur vs vertebra

BV/TV (%) BS/BV (1/mm)

Tb.Th (mm) Tb.Sp (mm)

0.136 -0.014

0.111 0.179

0.403 * 0.932 * 0.496 * 0.268 *

y= 23.752 + 0.156 x y= 45.286 - 0.0166 x y= 0.0754 + 0.0994 x y= 0.119 + 0.481x

Table 3: The pairs of BV/TV BS/BV, Tb.Th, Tb. N , Tb. Sp of the femoral and tibial ROIs with positive correlation coefficients and P values below 0.05 tend to increase together. No correlation was found between the femoral and vertebral regions.

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Dr. med.

Hansjoerg Heep

Korrespondenzadresse:

Department of Orthopaedics, University of Duisburg-Essen Pattbergstr. 1-3

D-45329 Essen

hansjoerg.heep@uni-due.de Phone.: 0201/4089-2147 Fax: 0201/4089-2722 Akademischer Lebenslauf

Oberarzt, Orthopädische Klinik der Universität Duisburg- Essen im Evangel. Krankenhaus Essen-Werden

1986-1988 Studium der Chemie Universität Heidel- berg

1988-1994 Studium der Humanmedizin Heidelberg und München (LMU, Sommersemester 1988)

1997 Dissertation „Quantifizierung der RES- Clearance nach Reperfusion bei Leber- transplantation unter klinischen Bedin- gungen“

2001 Facharzt Chirurgie

2003 Schwerpunkt Unfallchirurgie

2006 Facharzt Orthopädie und Unfallchirurgie 2007 Zusatzbezeichnung Spezielle Orthopädi- sche Chirurgie, Physikalische Therapie 2003 Oberarzt Unfallchir., Universität Düssel-

dorf

2006 Orthopädie Universität Duisburg-Essen

(8)

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