Different skeletal regional response to continuous brain infusion of leptin in the rat

Different skeletal regional response to continuous brain infusion of leptin in the rat

peptides 27 (2006) 1426–1433 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides Different skeletal regional resp...

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peptides 27 (2006) 1426–1433

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/peptides

Different skeletal regional response to continuous brain infusion of leptin in the rat F. Guidobono a,*, F. Pagani a, V. Sibilia a, C. Netti a, N. Lattuada a, D. Rapetti a, E. Mrak b, I. Villa b, F. Cavani c, L. Bertoni c, C. Palumbo c, M. Ferretti c, G. Marotti c, A. Rubinacci b a

Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Italy Bone Metabolic Unit, Scientific Institute San Raffaele Hospital, Milan, Italy c Department of Anatomy and Histology, University of Modena and Reggio Emilia, Italy b

article info

abstract

Article history:

This study was designed to evaluate whether or not continuous intracerebroventricular

Received 27 July 2005

infusion of leptin (1.5 mg/rat/24 h, for 28 days) produced different regional response on the

Received in revised form

skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content

13 October 2005

(BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no

Accepted 14 October 2005

changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of

Published on line 29 November 2005

both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the

Keywords:

leptin-treated group as compared to controls. The results demonstrate that long-term

Leptin

central infusion of leptin activates bone remodeling with a negative balance. Leptin induces

Planar bone mineral density

distinct responses in the different structure of bone and in the axial and appendicular

DXA

skeleton. # 2005 Elsevier Inc. All rights reserved.

Volumetric bone mineral density pQCT Osteocalcin Deoxypyridinoline

1.

Introduction

Leptin is a peptide hormone produced by the obese gene, secreted primarily by adipocytes, which limits food intake, increases energy expenditure and regulates body weight [9]. In addition to its role as a hormonal regulator of energy homeostasis, the peptide can influence bone metabolism through both a peripheral mechanism and a central hypothalamic pathway [34,5]. Leptin acts directly on bone marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes [35]. Leptin is secreted by osteoblasts in culture and its receptor mRNA is expressed in

these cells, thus strongly supporting leptin as a local factor modulating bone metabolism [26]. The peptide is also effective in increasing proliferation of isolated fetal rat osteoblasts [3]. Peripheral administration of leptin stimulates bone growth, increases bone mineral content (BMC) and bone density (BMD) in mice lacking the obese gene, ob/ob mice, [29]. Moreover, peripheral leptin administration is effective in reducing trabecular bone loss in ovariectomized rats [2], in reducing bone fragility in male mice [3] and in preventing disuseinduced bone loss in tail-suspended rats [17]. All these observations indicate a general consensus on a direct stimulatory effect of leptin on bone growth when adminis-

* Corresponding author. Tel.: +39 025 031 6997; fax: +39 025 031 6981. E-mail address: [email protected] (F. Guidobono). 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.10.014

peptides 27 (2006) 1426–1433

tered peripherally. Some controversy exists on the effect on bone metabolism in leptin-deficient or leptin-resistant animals. Ducy et al. [5] showed that knockout ob/ob mice and mice lacking a functional leptin receptor, db/db mice, have significantly greater bone mass than wild-type mice. In agreement, Schilling et al. [28] found that even obese Zucker rats, that lack a functional leptin receptor, have a high bone mass as observed by Ducy et al. [5] in ob/ob mice. On the contrary, Lorentzon et al. [16] as early as 1986 demonstrated that mice with genetic diabetes (db/db) are osteopenic. These results were confirmed by Steppan et al. [29] who showed that ob/ob mice have lower total body BMC and BMD as well as lower femoral BMC and BMD than normal mice. Bone mass controversies were also found in obese Zucker rats showing lower femoral BMC and BMD by DXA [8,18] or no significant differences by pQCT although a trend to a decrease in bone volume was observed as shown by a decrease in trabecular number and thickness and an increase in osteoclast surface [32]. The role of leptin in the control of bone mass involves different sites of action since intracerebroventricular (i.c.v.) injection of leptin decreases bone density in either wild type or leptin-deficient mice [5] through activation of its own receptors present in the hypothalamus [4]. Thus, leptin action is anabolic when administered peripherally, and yet produces osteopenic effects when administered centrally. Physiologically the net results of leptin effect on bone may be secondary to the combination of anabolic peripheral and catabolic central effects depending on serum leptin concentration and leptin central transport [10]. In fact, the peptide originates outside the central nervous system and it is transported into the brain by a saturable leptin transport system [12]. These contrasting evidences have led Hamrick et al. [11] to suggest that bone phenotype of leptin-deficient mice is more complex than previously appreciated. He has also demonstrated that response of bone to altered leptin signaling is not uniform throughout the skeleton but varies between axial and appendicular regions thus opening the need as suggested by Reid [24], of a reappraisal of the central regulation of bone physiology. The current study was designed to evaluate whether or not regional effects of leptin on the skeleton were also present following long-term treatment with the peptide injected i.c.v. in normal growing rats. Therefore, the effect of continuous i.c.v. infusion of leptin on bone was examined by measuring BMC and BMD by DXA and volumetric BMD by pQCT in the axial and appendicular skeleton. The markers of bone metabolism were also evaluated. The results show that i.c.v. leptin reduces trabecular bone in the appendicular and not in the axial regions of the skeleton and maintains the activation frequency of bone turnover of a faster growing state.

tiletamineHCl + zolazepamHCl (Zoletil 100, Virbac srl, Milano, Italy).

2.2.

Materials and methods

2.1.

Compounds

Leptin (Sigma, St. Louis, MO, USA) was dissolved in HCl 15 mM, buffered with NaOH 7.5 mM, and diluted in saline for the i.c.v. infusion by Alzet minipumps of 0.25 ml/h for a total daily dose of 1.5 mg/rat for 28 days. For anesthesia it was used

Animals and surgical procedure

Male Sprague–Dawley rats weighing 275–300 g were purchased from Charles River Laboratories (Calco, Italy). All rats were housed in single cages under controlled conditions (22  2 8C, 65% humidity, 12-h light:12-h dark cycle). Continuous central administration of leptin at a uniform rate was achieved via osmotic minipumps (Alzet, Model 2004, 0.25 ml/h; Alza, Palo Alto, CA, USA). Briefly, under anesthesia, rats were implanted with a 28-gauge stainless steel L-shaped cannula (ALZET Brain Infusion Kit; Alza) into the lateral brain ventricle. One end of the cannula was connected with a tube to an Alzet osmotic minipump inserted subcutaneously into the animal dorsum. Pumps are designed to deliver 0.25 ml/h at normal body temperature. All experiments were carried out according to the guidelines of the Animal Care Committee of the Department of Pharmacology of the University of Milan.

2.3.

Experimental protocol

Rats were allowed to acclimatize for a week and then were implanted with the Alzet minipumps system. After three days from surgery, rats were randomly divided into three groups: one group leptin-treated (1.5 mg/rat/24 h, infusion for 28 days) with food ad libitum; one group pair-fed and sham-operated, with food matched to that consumed by the leptin-treated group; one group of controls with food ad libitum. The amount of food administered to pair-fed rats was established by weighing the food consumed by the leptin-treated rats on a daily basis. Food intake and body weight of the rats were registered every day and every week respectively. Bone areas, bone mineral content (BMC) and bone mineral density (BMD) measured by DXA, were evaluated at the start of the experiment (t0) and after 28 days (t28) of treatment. Twenty-four hour urine samples for the measurement of deoxypyridinoline (DPD), a marker of bone resorption, were collected from rats housed in metabolic cages (Techniplast, Varese, Italy) at t0 and t28. Samples were immediately frozen and stored at 20 8C until assayed. Blood samples for the measurement of osteocalcin (OC), a marker of bone formation, were drawn at t0 and t28, under light ether anesthesia, by cardiac puncture. Plasma was stored at 80 8C until assayed. At the end of the experiment tibia and femur were excised for trabecular, cortical and sub cortical volumetric BMD (tvBMD) determinations by pQCT and for histomorphometric analysis.

2.4.

2.

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Biochemical analysis

Total urinary DPD levels were measured using an EIA kit (Quidel Corporation, San Diego, CA, USA). Intra- and interassay variations were 5.5 and 3.1%, respectively. The total daily excretion of DPD was corrected for creatinine excretion. Urinary creatinine was measured colorimetrically using a commercial kit (Quidel Corporation). Total serum OC was determined using a commercial immunoenzymatic kit (Biomedical Technologies Inc.,

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Stoughton, MA, USA). The intra-assay variation was 4%, while the inter-assay variation was 7%. All biochemical parameters are expressed as percentage variation versus basal values.

2.5.

Dual energy X-ray absorptiometry (DXA)

All rats were anaesthetized with Zoletil (40 mg/kg, i.m.) and scanned with Hologic QDR-1000 instrument (Hologic Inc., Waltham, MA, USA) in the ultra-high resolution mode with a longitudinal line spacing of 0.254 mm, implemented with a collimator 1.0 mm in diameter and with the High Resolution Software (version 4.47) adapted for small animals. Three regions of interest were chosen: the entire femur, the femoral metaphysis and the lumbar vertebrae (L1–L4). The software provided the total area (cm2) of the planar image of the selected segments, the BMC in mg and the BMD in mg/cm2. Coefficients of variation were 3% for BMC and 1% for BMD. The precision and accuracy of DXA in small laboratory animals has been widely validated [21,1].

2.6.

means of percentage at t28 versus basal level (t0). Data were analyzed by one-way ANOVA followed by Dunnett’s test for multiple comparison. Changes from baseline of the biochemical markers, were analyzed by paired Student’s t-tests. Comparisons between groups at t28 expressed in percentage from basal were analyzed by a two-tailed unpaired Student’s ttests for non-parametric data. A probability of p < 0.05 was considered significant.

3.

Results

The continuous i.c.v. infusion of leptin decreased food intake compared to controls up to 16 days, after that time all animals ate normally (Fig. 1A). The lower food consumption was associated with a lower body weight that was statistically significant ( p < 0.001) in the leptin group and not in the pair fed animals that showed a lower growth curve that, however, did not reach statistical significance (Fig. 1B).

Peripheral quantitative computed tomography (pQCT)

The left femurs, tibiae and the third lumbar vertebrae of rats were scanned with a peripheral quantitative computed tomography (pQCT) system, the Stratec SA-plus (Stratec Medizintechnik GmbH, Pforzheim, Germany), using a voxel size of 70 mm3. The distal metaphysis of femurs were scanned adjusting the scan line to 5 mm proximal to the distal end of femur using the scout view property of the pQCT software. The proximal metaphysis of the tibiae were scanned adjusting the scan line 4.5 mm distal to the proximal end of the tibiae. The scans of femurs and tibiae were performed with the peel mode 2 and the contour mode 2 with inner threshold of 395 mg/cm3. The vertebrae were scanned adjusting the scan line to the middle of the vertebral body with the peel mode 20 and the contour mode 2 for high resolution scans.

2.7.

Histomorphometry

Three controls, three pair-fed and three leptin-infused rats were intraperitoneally injected with oxitetracycline (40 mg/kg) on the 6th and the 26th day of the experimental period, and were sacrificed on the 28th day. Periosteal and endosteal appositional growth was measured on four serial crosssections, methylmethacrylate embedded, taken from middiahyseal levels of the right tibia in all rats. The following parameters were measured on each section by means of VIDAS Zeiss image analyzer: - total cortical bone area (CA), excluding diaphyseal canal; - the sum of the areas (SA) of bone between the two tetracycline labels at the periosteal and endosteal levels, respectively.

2.8.

Statistical analysis

Statistical analysis was performed by a statistical package (PRISM, vers. 2.01 GraphPad Software San Diego, CA, USA). Data are shown as the means of the D(t28 t0)  S.E.M. or as

Fig. 1 – (A) Food intake and (B) body weight of leptin-treated animals (1.5 mg/rat/24 h, i.c.v. infusion for 28 days), pairfed rats and controls with access to food ad libitum. Values are the mean W S.E.M. of nine to seven rats per group. a p < 0.05; bp < 0.01; cp < 0.001; compared with controls, ANOVA and Dunnett test.

peptides 27 (2006) 1426–1433

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Table 1 – Effect of leptin (1.5 mg/rat/24 h) i.c.v. infusion for 28 days on total femural area, BMC and planar BMD measured at the start (t0) and at the end (t28) of the experiment by DXA Area (cm2) t0

t28

Controls Pair-fed Leptin

1.422  0.019 1.478  0.012 1.441  0.015

1.778  0.058 1.758  0.032 1.748  0.022

0.354  0.051 0.278  0.022 0.306  0.031

BMC (mg) Controls Pair-fed Leptin

329.1  11.0 343.7  7.0 342.1  9.0

520.4  17.0 509.9  13.0 495.6  5.7

191.2  14.7 166.3  12.5 153.6  10.4*

292.9  5.1 290.1  4.1 283.7  3.1

61.8  4.0 57.7  5.5 46.5  2.0**

BMD (mg/cm2) Controls 231.1  5.4 Pair-fed 232.4  4.0 Leptin 237.2  4.6

D(t28

t0)

Data are expressed as mean  S.E.M. (n = 7–9 group, ANOVA and Dunnett test). D(t28 t0) = changes observed in the 28 days of the experiment. * p < 0.05 vs. control. ** p < 0.01 vs. control.

Despite the fall in body weight no changes in total femoral areas were detected between leptin treated and control groups as measured by DXA. The natural increment with age of planar femoral BMC and BMD was lower ( p < 0.05, p < 0.01) in the group of animals treated with leptin than in control and pairfed animals (Table 1). This variation could be explained by the significant ( p < 0.05) reduction of the trabecular bone BMC and BMD in the femoral metaphysis (Fig. 2A and B), as no changes were detected in the femoral diaphysis (Table 2). In Table 2 are also reported the data of areas, BMC and BMD of the lumbar vertebrae (L1–L4) that showed no statistical differences between leptin treated and control or pair-fed rats. At the end of the experiment (t28) excised vertebrae, femurs and tibiae were examined by pQCT. Total area, trabecular area and cortical and sub cortical areas as well as the volumetric BMD of the trabecular bone and of the cortical bone were analyzed (Table 3). In agreement with the results obtained with DXA in the vertebrae, also the results with pQCT showed no differences between the groups in all these parameters. Cortical areas and volumetric trabecular BMD resulted significantly decreased in leptin-treated animals in femur ( p < 0.05) and in tibia ( p < 0.01) compared with controls. Cortical volumetric BMD was found to be slightly but significantly ( p < 0.05) increased in the tibia of pair-fed animals compared to controls. This finding could be consequent to the slight decrease (but not significant) of the tibial area. The length of tibiae was also measured and no statistical differences were detected amongst the groups (data not shown). Continuous long-term brain infusion of leptin was able to modify the biochemical markers of bone metabolism (Figs. 3 and 4). Serum OC in the control group decreased ( p < 0.001) of 38.9% at t28 compared to the basal level (t0), as the animals reached a steady state of bone maturity. Similarly in pair-fed animals the decrement was of 23.4% ( p < 0.01). In the leptintreated animals the decrease was 14.4% ( p < 0.05) versus basal level. This reduction was significantly lesser ( p < 0.05) than

Fig. 2 – (A) Bone mineral content and (B) planar bone mineral density measured at femoral metaphysis in leptin-treated animals (1.5 mg/rat/24 h i.c.v. infusion for 28 days), pair-fed rats and controls with access to food ad libitum. Data are expressed as the differences between t28 and t0. Values are the mean W S.E.M. of nine to seven rats per group. *p < 0.05 compared with controls, ANOVA and Dunnett test.

that observed in controls (Fig. 3). Urine excretion of DPD after 28 days decreased of 32.5% ( p < 0.01) in the control group and 32% ( p < 0.05) in pair-fed rats compared with basal value. In leptin-treated animals DPD excretion did not decrease and remained similar to that at basal level ( 3.2%), significantly different ( p < 0.01) compared to controls (Fig. 4). The data of the histomorphometry analysis of periosteal and endosteal appositional growth, measured with double tetracycline labeling (Fig. 5), are reported in Table 4. No significant differences were observed among the three groups of rats, though bone growth shows a tendency to decrease in the leptin-treated group.

4.

Discussion

These studies show that i.c.v. administration of leptin for 28 days is associated with regional and structural differences in bone mineral accretion in growing rats. In fact BMC and planar BMD by DXA were lower in leptin-treated animals than controls and pair-fed animals. The lower mineral acquisition was only at the expenses of metaphyseal sites of the appendicular skeleton, mostly trabecular. Diaphyseal sites

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Table 2 – BMC and planar BMD of femoral diaphysis and vertebrae of rats treated i.c.v. with leptin (1.5 mg/rat/24 h, infusion for 28 days) measured by DXA at the start (t0) and at the end (t28) of the experiment Femoral diaphysis

BMD (mg/cm2)

BMC (mg) t0

t28

Controls Pair-fed Leptin

59.64  2.17 60.04  0.70 59.30  1.12

84.32  3.60 87.62  2.10 81.29  1.20

Vertebrae Controls Pair-fed Leptin

337.9  17.40 339.2  14.88 340.3  15.16

489.8  29.6 493.8  24.6 512.6  16.0

to

t28

24.68  2.92 27.58  1.59 21.99  1.68

276.2  8.68 274.0  2.89 275.2  4.83

346.0  8.9 349.0  5.0 337.5  3.5

70.04  4.6 75.04  7.2 62.30  5.5

151.9  25.9 154.6  16.9 172.3  15.6

194.3  7.9 187.1  6.5 194.9  5.5

226.9  7.5 223.1  9.2 235.1  4.2

32.56  7.01 36.04  4.92 40.16  4.58

D(t28

t0)

Data are expressed as mean  S.E.M. (n = 7–9 rats/group, ANOVA and Dunnett test); D(t28 experiment.

and vertebrae were not affected. The site and structure specific effects of i.c.v. leptin infusion were confirmed by pQCT analysis, being the volumetric BMD lower only in the trabecular tibia and femur and not in the vertebrae. This finding was also confirmed in the tibia by the lack of changes in cortical periosteal and endosteal bone growth, measured with double tetracycline labeling. The bone changes observed in leptin-treated rats compared to controls or pair-fed animals were associated with a different secretion profile of the biochemical markers of bone remodeling. In fact in leptintreated rats OC and DPD remained at the same elevated levels as those detected at the start of the experiment, that is, animals in rapid growth [25]. The regional and structural differences in the bone mineral accretion in growing rats under central leptin excess, here outlined, is in line with the finding of Hamrick et al. [11] that showed that the effects of altered leptin signaling (ob/ob mice) on bone are not uniform throughout the skeleton. In the lumbar spine, bone shows higher BMC, BMD and trabecular bone volume while cortical bone shows a reduced thickness. In the femur instead, bone shows reduced BMC and BMD and lower trabecular bone volume. In the lumbar vertebrae there was no increase of adipocytes [11]. In the femur of ob/ob mice instead,

D(t28

t0)

t0) = changes observed in the 28 days of the

there was increased number of marrow adypocytes and decreased osteocyte population that could have inhibited the osteoblasts activity [19]. As suggested by Reid [24] this contrasting response of marrow cells in the spine and femur of leptin-deficient mice may in part explain the differences in bone mass and density observed between the femur and lumbar spine of ob/ob mice. Thus leptin deficiency has very different effects on the axial and appendicular skeleton as in the condition of central leptin excess; there is a reduction in trabecular density specific of the appendicular skeleton, and not in the axial skeleton that was not affected. This regional and structural difference under both leptin deficiency and excess has opened a new perspective in evaluating the centrally mediated skeletal effect of leptin. Classically, it was ascribed to an inhibitory action on the osteoblast activity, like a ‘‘brake’’ on the bone anabolic action of hormones, leptin included. Leptin may instead be one of the key factors linking energy availability to bone metabolism, as pointed out by Reid [23]. A proper balance of bone mass in response to nutritional status can be achieved due to the dual action of central and peripheral leptin on bone, as reviewed and outlined by several authors [36,13,7]. This study has demonstrated that central leptin excess in growing rats is associated with the maintenance of the

Table 3 – Volumetric pQCT analysis of rat bones after 28 days i.c.v. infusion with leptin (1.5 mg/rat/24 h) Area (mm2)

BMD (mg/cm3)

Total

Trabecular

Cortical

Trabecular

Cortical

Femurs Controls Pair-fed Leptin

27.48  0.95 27.53  0.81 24.49  1.2

9.56  0.55 9.51  0.58 10.98  0.75

17.91  0.90 18.01  1.02 13.54  1.21*

230.1  9.01 229.7  7.53 188.1  12.92*

666.4  14.95 670.0  14.58 728.8  51.56

Tibiae Controls Pair-fed Leptin

24.20  1.10 20.76  0.61 21.08  0.64

13.53  0.67 11.54  0.47 12.91  0.53

10.66  0.59 9.20  0.28 8.16  0.23**

166.4  14.16 134.3  11.12 114.0  6.92**

823.4  20.82 904.0  26.05* 873.3  12.65

Vertebrae Controls Pair-fed Leptin

31.92  1.43 34.45  1.04 33.32  1.31

14.69  0.49 15.50  0.55 14.99  0.59

17.95  0.60 18.95  0.66 18.33  0.72

186.98  10.39 172.20  6.36 174.98  7.73

839.03  33.00 831.53  10.30 798.27  14.83

Data are the mean  S.E.M. of 7–9 rats/group (ANOVA and Dunnett test). p < 0.05 vs. control. ** p < 0.01 vs. control. *

peptides 27 (2006) 1426–1433

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Fig. 3 – Serum osteocalcin levels in leptin-treated animals (1.5 mg/rat/24 h infusion for 28 days), pair-fed rats and controls with access to food ad libitum. Columns represent data expressed in percentage vs. basal levels detected at t0. *p < 0.05; **p < 0.01; ***p < 0.001 compared with basal levels, Student’s t-test for paired observation. p < 0.05 compared with controls, ANOVA and Dunnett test. n = 9–7 rats/group.

Fig. 4 – Urinary excretion of deoxypyridinoline in leptintreated animals (1.5 mg/rat/24 h infusion for 28 days), pairfed rats and controls with access to food ad libitum. Data are expressed as a ratio to 24 h creatinine urinary excretion. Columns represent the changes in percentage vs. basal levels (t0). *p < 0.05; **p < 0.01; compared with t0, Student’s t-test for paired observation. p < 0.01 compared with controls ANOVA and Dunnett test. n = 9–7 rats/group.

activation frequency of bone remodeling to a faster growing rate. This implies that the effect of central leptin cannot be ascribed only to the inhibitory activity on osteoblasts because in this case, OC secretion would have been inhibited. This study instead failed to demonstrate a reduced OC secretion, which was maintained at higher level than in controls, indicating that osteoblast activity was not

inhibited. This is also supported by cortical bone appositional growth that was not significantly different from controls. The observed elevated urinary excretion of DPD, is consistent with a stimulatory action on bone resorption that could be responsible for the negative bone balance at the appendicular skeleton produced by centrally injected leptin.

Fig. 5 – Micrographs under UV light of cross-sections at the mid-diaphyseal level of the right tibia in control (A), pair-fed (B) and leptin-infused (C) rats.

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Table 4 – Mean values W S.D. of CA and SA parameters, expressed in mm2, evaluated from four cross-sections at mid-diaphyseal levels of the right tibia in rats of each group

Controls Pair-fed Leptin

CA

SA

% SA/CA

4.87  0.26 4.69  0.24 4.24  0.28

0.82  0.08 0.66  0.07 0.53  0.07

16.8  3.8 14.1  1.2 12.5  0.8

No significant differences were detected among the three groups of rats, though bone appositional growth shows a tendency to decrease in leptin infused rats. CA is the total cortical bone area, excluding diaphysial canal; SA, the sum of the areas of bone between the two tetracycline labels at periosteal and endosteal site; % SA/CA, the percentage of newly formed bone with respect to total bone.

It is possible that the effect of leptin on bone remodeling could also involve the activity of growth hormone (GH). Leptin is in fact a potent stimulator of both spontaneous pulsatile GH secretion and of the GH response to GHRH [33]. It is well known that GH is among the major regulators of both bone formation and resorption. Interestingly, the action of GH on bone metabolism follows a biphasic model. Initially GH treatment results in a phase of increased bone resorption with increased number of bone-remodeling units and a concomitant bone loss that is then followed by a phase of increased bone formation [20]. In addition the regional bone effects and the preservation of the bone remodeling to a faster growing state might be ascribed to the effects exerted by leptin on several nervous relay systems mediated by the hypothalamic ventromedial nuclei via the sympathetic nervous system (SNS) [31,27], considered the downstream signal of the centrally mediated effects of leptin on bone. In particular it has been reported that mice lacking the b2-adrenergic receptors are resistant to the bone-reducing effect exerted by leptin in the CNS [6]. It could be assumed that the heterogeneity of bone loss under central leptin excess could be ascribed to differences in b2-adrenergic receptor signaling on osteoblasts. Sympathetic pathway stimulation favors bone resorption by increasing the expression of RANKL, the principal physiological inducer of osteoclast formation [6]. Differences in the neural fibers innervating specific regions of the skeleton and in the local density of the receptors, could account for the disparate responses to leptin [24]. It is intriguing the observed disparity between cortical and trabecular responses of bone to leptin treatment within the same skeletal region. It could be speculated that the compartmental effect of leptin-SNS activation is the result of a higher availability of osteoclast precursors owing to the greater surface of the cancellous bone (versus cortical) exposed to the red marrow [22]. The regional difference in the effect of leptin on bone might also be related to the complex interplay between bone mechanical competence and the central neurogenic control of skeletal homeostasis. In fact, SNS by mediating the bone remodeling effects of different loading/unloading regimen [14] is considered one of the major transmitter pathways of mechanical loading in rat bone [15]. Since an intact SNS is required for the action of central leptin on bone remodeling [6], it follows that under central leptin excess the activation of the SNS induces bone

loss in the methaphyseal trabeculae without major impairment of mechanical competence, being cortical compartment unaffected. It should be ruled out that the negative bone effect of leptin is secondary to reduced food intake since in the pair-fed animals, bone mass was not significantly affected. Accordingly it has been shown that leptin anorexigenic and antiosteogenic actions are mediated by different hypothalamic nuclei [30]. Leptin had no significant effects on bone growth or bone size as no significant differences in the bone length or in the bone volume were observed by DXA or by pQCT. At difference the peripheral administration of leptin in animals lacking the leptin gene or the leptin receptor gene led to a significant increase in femoral length and total body bone area [29]. However the actual experimental conditions, central versus peripheral administration of the peptide, and the inborn defect of leptin-deficient mice, cannot be compared. In conclusion, this study has demonstrated that central long-term infusion of leptin leads to regional and structural differences in the skeletal responses. The trabecular compartments of the appendicular skeleton showed a decrease in bone mass accrual that was not apparent in cortical compartment or in the axial skeleton. The effect involves the preservation of bone remodeling at the activation frequency level of a faster growing state.

Acknowledgment This work was supported by funds from the Italian Ministry of University and Research, Cofin2002 and FIRB2001.

references

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