Different skeletal regional response to continuousbrain 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

aDepartment of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, ItalybBone Metabolic Unit, Scientific Institute San Raffaele Hospital, Milan, ItalycDepartment of Anatomy and Histology, University of Modena and Reggio Emilia, Italy

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 3

a r t i c l e i n f o

Article history:

Received 27 July 2005

Received in revised form

13 October 2005

Accepted 14 October 2005

Published on line 29 November 2005

Keywords:

Leptin

Planar bone mineral density

DXA

Volumetric bone mineral density

pQCT

Osteocalcin

Deoxypyridinoline

a b s t r a c t

This study was designed to evaluate whether or not continuous intracerebroventricular

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

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

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

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

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

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

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

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

skeleton.

# 2005 Elsevier Inc. All rights reserved.

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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 hypotha-

lamic 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

* Corresponding author. Tel.: +39 025 031 6997; fax: +39 025 031 6981.E-mail address: francesca.guidobono@unimi.it (F. Guidobono).

0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2005.10.014

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 disuse-

induced 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-

.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 3 1427

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 sig-

nificantly greater bone massthan wild-type mice. Inagreement,

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. Physiologi-

cally 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.

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

tiletamineHCl + zolazepamHCl (Zoletil 100, Virbac srl, Milano,

Italy).

2.2. Animals and surgical procedure

Male Sprague–Dawley rats weighing 275–300 g were pur-

chased 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). Contin-

uous 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. Biochemical analysis

Total urinary DPD levels were measured using an EIA kit

(Quidel Corporation, San Diego, CA, USA). Intra- and inter-

assay 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.,

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 31428

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. 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 cross-

sections, methylmethacrylate embedded, taken from mid-

diahyseal levels of the right tibia in all rats. The following

parameters were measured on each section by means of

VIDAS Zeiss image analyzer:

- t

otal cortical bone area (CA), excluding diaphyseal canal;

- t

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), pair-

fed rats and controls with access to food ad libitum. Values

are the mean W S.E.M. of nine to seven rats per group.ap < 0.05; bp < 0.01; cp < 0.001; compared with controls,

ANOVA and Dunnett test.

he 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

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 biochem-

ical 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 t-

tests 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).

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 3 1429

Table 1 – Effect of leptin (1.5 mg/rat/24 h) i.c.v. infusion for28 days on total femural area, BMC and planar BMDmeasured at the start (t0) and at the end (t28) of theexperiment by DXA

Area (cm2)

t0 t28 D(t28 � t0)

Controls 1.422 � 0.019 1.778 � 0.058 0.354 � 0.051

Pair-fed 1.478 � 0.012 1.758 � 0.032 0.278 � 0.022

Leptin 1.441 � 0.015 1.748 � 0.022 0.306 � 0.031

BMC (mg)

Controls 329.1 � 11.0 520.4 � 17.0 191.2 � 14.7

Pair-fed 343.7 � 7.0 509.9 � 13.0 166.3 � 12.5

Leptin 342.1 � 9.0 495.6 � 5.7 153.6 � 10.4*

BMD (mg/cm2)

Controls 231.1 � 5.4 292.9 � 5.1 61.8 � 4.0

Pair-fed 232.4 � 4.0 290.1 � 4.1 57.7 � 5.5

Leptin 237.2 � 4.6 283.7 � 3.1 46.5 � 2.0**

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.

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.

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 pair-

fed 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

andcortical and subcortical areasaswell as the volumetricBMD

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 leptin-

treated animals the decrease was 14.4% (p < 0.05) versus basal

level. This reduction was significantly lesser (p < 0.05) than

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

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 31430

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, infusionfor 28 days) measured by DXA at the start (t0) and at the end (t28) of the experiment

Femoral diaphysis BMC (mg) BMD (mg/cm2)

t0 t28 D(t28 � t0) to t28 D(t28 � t0)

Controls 59.64 � 2.17 84.32 � 3.60 24.68 � 2.92 276.2 � 8.68 346.0 � 8.9 70.04 � 4.6

Pair-fed 60.04 � 0.70 87.62 � 2.10 27.58 � 1.59 274.0 � 2.89 349.0 � 5.0 75.04 � 7.2

Leptin 59.30 � 1.12 81.29 � 1.20 21.99 � 1.68 275.2 � 4.83 337.5 � 3.5 62.30 � 5.5

Vertebrae

Controls 337.9 � 17.40 489.8 � 29.6 151.9 � 25.9 194.3 � 7.9 226.9 � 7.5 32.56 � 7.01

Pair-fed 339.2 � 14.88 493.8 � 24.6 154.6 � 16.9 187.1 � 6.5 223.1 � 9.2 36.04 � 4.92

Leptin 340.3 � 15.16 512.6 � 16.0 172.3 � 15.6 194.9 � 5.5 235.1 � 4.2 40.16 � 4.58

Data are expressed as mean � S.E.M. (n = 7–9 rats/group, ANOVA and Dunnett test); D(t28 � t0) = changes observed in the 28 days of the

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 leptin-

treated 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,

Table 3 – Volumetric pQCT analysis of rat bones after 28 days

Area (mm2)

Total Trabecular

Femurs

Controls 27.48 � 0.95 9.56 � 0.55

Pair-fed 27.53 � 0.81 9.51 � 0.58

Leptin 24.49 � 1.2 10.98 � 0.75

Tibiae

Controls 24.20 � 1.10 13.53 � 0.67

Pair-fed 20.76 � 0.61 11.54 � 0.47

Leptin 21.08 � 0.64 12.91 � 0.53

Vertebrae

Controls 31.92 � 1.43 14.69 � 0.49

Pair-fed 34.45 � 1.04 15.50 � 0.55

Leptin 33.32 � 1.31 14.99 � 0.59

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.

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 massand density observedbetween 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

trabeculardensity specific of theappendicularskeleton,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

i.c.v. infusion with leptin (1.5 mg/rat/24 h)

BMD (mg/cm3)

Cortical Trabecular Cortical

17.91 � 0.90 230.1 � 9.01 666.4 � 14.95

18.01 � 1.02 229.7 � 7.53 670.0 � 14.58

13.54 � 1.21* 188.1 � 12.92* 728.8 � 51.56

10.66 � 0.59 166.4 � 14.16 823.4 � 20.82

9.20 � 0.28 134.3 � 11.12 904.0 � 26.05*

8.16 � 0.23** 114.0 � 6.92** 873.3 � 12.65

17.95 � 0.60 186.98 � 10.39 839.03 � 33.00

18.95 � 0.66 172.20 � 6.36 831.53 � 10.30

18.33 � 0.72 174.98 � 7.73 798.27 � 14.83

.

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 3 1431

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 leptin-

treated animals (1.5 mg/rat/24 h infusion for 28 days), pair-

fed 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

Fig. 5 – Micrographs under UV light of cross-sections at the mid

and leptin-infused (C) rats.

inhibited. This is also supported by cortical bone apposi-

tional 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.

-diaphyseal level of the right tibia in control (A), pair-fed (B)

p e p t i d e s 2 7 ( 2 0 0 6 ) 1 4 2 6 – 1 4 3 31432

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

CA SA % SA/CA

Controls 4.87 � 0.26 0.82 � 0.08 16.8 � 3.8

Pair-fed 4.69 � 0.24 0.66 � 0.07 14.1 � 1.2

Leptin 4.24 � 0.28 0.53 � 0.07 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 expres-

sion 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 compe-

tence and the central neurogenic control of skeletal home-

ostasis. 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 impair-

ment 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. Accord-

ingly it has been shown that leptin anorexigenic and

antiosteogenic actions are mediated by different hypothala-

mic 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 in-

born 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 compart-

ments 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.

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