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Endocrinology, doi:10.1210/en.2007-0171
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Endocrinology Vol. 148, No. 7 3111-3121
Copyright © 2007 by The Endocrine Society

Dlk1/FA1 Is a Novel Endocrine Regulator of Bone and Fat Mass and Its Serum Level Is Modulated by Growth Hormone

Basem M. Abdallah, Ming Ding, Charlotte H. Jensen, Nicholas Ditzel, Allan Flyvbjerg, Thomas G. Jensen, Frederik Dagnæs-Hansen, Jürg A. Gasser and Moustapha Kassem

Department of Endocrinology (Clinic for Molecular Endocrinology Treatment Laboratory) (B.M.A., M.D., N.D., M.K.) and Orthopaedic Research Laboratory, Department of Orthopaedics (M.D.), Odense University Hospital, DK-5000 Odense, Denmark; Department of Immunology and Microbiology (C.H.J.), University of Southern Denmark, DK-5230 Odense, Denmark; The Medical Research Laboratories and Medical Department of Diabetes and Endocrinology (A.F.) and Medical Microbiology and Immunology (F.D.-H.), Aarhus University Hospital, DK-8000 Aarhus, Denmark; Kennedy Institute (T.G.J.), National Eye Clinic, DK-2600 Glostrup, Denmark; and Novartis Institutes for BioMedical Research (J.A.G.), Pharma AG, CH-4002 Basel, Switzerland

Address all correspondence and requests for reprints to: Basem M. Abdallah, Ph.D., Department of Endocrinology and Metabolism, Odense University Hospital, Medical Biotechnology Center, University of South Denmark, DK-5000 Odense C, Denmark. E-mail: babdallah{at}health.sdu.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fat and bone metabolism are two linked processes regulated by several hormonal factors. Fetal antigen 1 (FA1) is the soluble form of dlk1 (delta-like 1), which is a member of the Notch-Delta family. We previously identified FA1 as a negative regulator of bone marrow mesenchymal stem cell differentiation. Here, we studied the effects of circulating FA1 on fat and bone mass in vivo by generating mice expressing high serum levels of FA1 (FA1 mice) using the hydrodynamic-based gene transfer procedure. We found that increased serum FA1 levels led to a significant reduction in total body weight, fat mass, and bone mass in a dose-dependent manner. Reduced bone mass in FA1 mice was associated with the inhibition of mineral apposition rate and bone formation rates by 58 and 72%, respectively. Because FA1 is colocalized with GH in the pituitary gland, we explored the possible modulation of serum FA1 by GH. Serum levels of IGF-I and IGF binding proteins did not change in FA1 mice, whereas increasing serum GH in normal mice using hydrodynamic-based gene transfer procedure dramatically reduced serum FA1 levels by 60%. Conversely, serum FA1 was increased 450% in hypophysectomized mice, and this high level was reduced by 40% during GH treatment. In conclusion, our data identify the FA1 as a novel endocrine factor regulating bone mass and fat mass in vivo, and its serum levels are regulated by GH. FA1 thus provides a novel class of developmental molecules that regulate physiological functions of the postnatal organisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A BODY OF RECENT studies have suggested that bone mass and fat mass are strongly associated entities. Several cross-sectional and longitudinal studies in adults and children have demonstrated that bone mass is positively correlated to body weight and fat mass (1). Also, low body weight and recent weight loss increases the risk for osteoporotic fractures (2, 3), whereas obesity seems to be protective (4). Furthermore, several histomorphometric studies of iliac crest biopsies have demonstrated increased bone marrow adipose tissue with aging and in osteoporotic women, and this was associated with decreased bone mass (5). At the cellular level, osteoblasts and adipocytes are derived from a common precursor mesenchymal stem cell (MSC) that resides in bone marrow and also in other connective tissue compartments (6). Several in vitro studies have also demonstrated the possible interconversion between the osteoblastic and adipocytic phenotypes depending on culture conditions (7).

The close link between bone and fat suggests the presence of common regulatory mechanisms. Several hormonal factors, including estrogen (8), insulin (9), and GH (10), are known to regulate bone mass and fat mass. In addition, several cytokines and tissue-derived factors have been identified for having hormone-like regulatory effects on both bone and fat mass, e.g. leptin (11, 12) and IL-6 (13).

We recently identified delta-like 1/fetal antigen 1 (dlk1/FA1) as a novel factor with regulatory effects on both osteoblast and adipocyte differentiation of human MSC (hMSC) (14). Dlk1/FA1 [also named preadipocyte factor 1 (pref-1)] is an imprinted paternally expressed gene that encodes for a transmembrane protein with six epidermal growth factor-like repeats in its extracellular domain similar to other members of the Notch/Delta/Serrate family. The importance of dlk1 in normal biology has been demonstrated in mice models and human diseases characterized by either deficiency or overexpression of Dlk1/FA1 (15, 16). These conditions are associated with a variety of developmental defects, including growth changes, changes in fat mass, and skeletal malformations (16, 17). In addition, in a variety of cellular models, dlk1/FA1 has been established as an inhibitor of adipocyte differentiation and plays a role in determining cell fate decision in many differentiation processes, including pancreatic islet cell differentiation (18), muscle cell (19), hepatocyte (20), and neuronal cell differentiation (21). Also, in bone marrow microenvironment, the expression of dlk1 by stromal cells is important for their supportive function of hematopoietic stem cell self-renewal and differentiation (22, 23). dlk1/FA1 can function as either a membrane-bound (dlk1) or soluble circulating protein (FA1) (24). FA1 is generated by proteolytic cleavage of the dlk1 extracellular domain by ADAM17/TACE (a disintegrin and metalloproteinase domain 17/TNF-{alpha} converting enzyme) (25). FA1 is found in most biological fluids, including urine, serum, amniotic fluid, and cord serum (26). It is present at very high levels in serum/amniotic fluid of pregnant human and mice, and its levels are decreased by 100-1000 times in adults (26, 27). Dlk1/FA1 is broadly expressed by most embryonic and fetal tissues in human and mouse, and its expression is postnatally down-regulated to be localized only to the hormone-secreting cells in the pituitary gland, pancreatic islets, adrenal glands, and testis (28, 29, 30) as well as in monoaminergic neurons in the central nervous system (4). Thus, whereas the local tissue production of dlk1/FA1 is limited in adults, all tissues are exposed to circulating FA1, which in turn may mediate biological functions that differ from the above mentioned developmental effects.

Thus, to investigate the systemic effects of the soluble circulating FA1 on the bone and fat metabolism in adult organisms, we generated mice expressing high serum levels of FA1 protein, using the systemic hydrodynamic gene transfer procedure (HGTP). Interestingly, high serum FA1 levels reduced both fat and bone mass in a dose-dependent manner. The detailed skeletal analysis revealed a marked effect of FA1 on reducing trabecular bone parameters and inhibiting bone formation rate (BFR). Furthermore, FA1 was shown to act downstream of GH, and its serum levels were inversely correlated with changes in serum levels of GH under pathophysiological conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental procedures were approved by the animal care and use committees of the University of South Denmark. BALB/cA (The Jackson Laboratory, Bar Harbor, ME) were bred and housed in standard conditions on a 12-h light, 12-h dark cycle at the animal facility. Male mice (8 wk old) were used in the study. The animal experiments were approved by the Danish Animal Experiment Inspectorate. Generation of hyphysectomized mice (HX mice) and HX mice injected with hGH construct have been described previously (31). The mice were housed three or four per cage and fed regular chow and water ad libitum. All mice were kept under humane conditions according to the regulations of the Home Office Animals (scientific procedures) Act 1986.

Expression vectors
For generating mice with high serum FA1 levels (FA1 mice), the full length of mouse Dlk1/Pref-1 cDNA (1118 bp) was subcloned into the EcoRI site downstream the human ubiquitin-C promotor of the expression plasmid PHD184 (32, 33) to produce plasmid named pUC-UBI-mDlk1. The previously described pUC-UBI-hGH plasmid (33) was used for generating mice with either high (hGHhigh mice) or different (hGHdiff mice) serum levels of hGH.

HGTP
HGTP was performed as described previously (34). In brief, animals were kept at a high ambient temperature to dilate the tail veins before the treatment. Immediately before injection, the animals were anesthetized with 4% (vol/vol) halothane air until digital reflex was absent. The gas inhalation anesthesia was used for fast induction and fast recovery. Naked DNA plasmid was administered to the animals by injecting into the tail vein 2.2–2.6 ml sterile saline solution (in mM: 147 NaCl, 4 KCl, and 1.13 CaCl2) corresponding to 8% of the body weight and containing 22.73 µg pUC-UBI-mDlk1 DNA per milliliter in the case of FA1 mice, 20–50 µg pUC-UBI-hGH plasmid in the case of hGHdiff mice, or 75 µg pUC-UBI-hGH plasmid in the case of hGHhigh mice. Male age-matched control group received saline solution. The injection was performed within 5–7 sec for all groups. After the injection, the animals were allowed to recover with the ambient temperature raised to approximately 28 C. For the FA1 mice group, injection was performed every 2 wk over a period of 2 months, and FA1 serum levels were determined 1 wk after each injection to ensure maintaining the circulated FA1 at high levels during the entire experiment period. Control mice group received similar number of injections with saline solution. For both hGHhigh mice and hGHdiff mice groups, one single injection was shown to achieve high and prolonged expression of GH over a period of more than 6 months (33).

Detecting the expression of mouse Dlk1 (mDlk1) staining for mdlk1/FA1
To confirm the localization and expression of mDlk1 plasmid in the liver of the mDlk1-injected mice, paraffin-embedded sections from different tissues were prepared and stained with a rabbit anti-mFA1 antibody as described previously (35). The staining procedure was performed on an automated immunostainer (TechMate 1000; DakoCytomation, Glostrup, Copenhagen) using the biotin-streptavidin detection system (ChemMate-HRP/DAB; DakoCytomation) or manually using 3-amino-9-ethylcarbazol as chromogen. Epithelial cells were identified in consecutive parallel sections with monoclonal antibodies to cytokeratin (CAM 5.2; Beckton Dickinson, Orangeburg, NY), and corresponding sections were stained with hematoxylin and eosin.

FA1 ELISA
Blood samples were collected from the retro-orbital plexus. The serum FA1 level was measured using a previously described sandwich ELISA technique using immunospecifically purified rabbit anti-mFA1 as catcher and biotinylated rabbit anti-mFA1 as indicator (27). The FA1 expression was represented as nanograms of FA1 per milliliter of serum.

Serum IGF-I and IGF binding protein (IGFBP) measurements
Serum IGF-I levels were measured by RIA using a polyclonal rabbit antibody (Nichols Institute Diagnostics, San Capistrano, CA) and recombinant human IGF-I as standard (GE Healthcare, Little Chalfont, UK). Monoiodinated IGF-I (125I-labeled [Tyr31]IGF-I) was obtained from Novo-Nordisk (Bagsvaerd, Denmark). Serum IGFBPs were measured by Western ligand blotting assay as described previously (36).

hGH measurement
The serum hGH concentration was measured by a commercial noncompetitive time-resolved immunofluorometric hGH assay (TR-IFMA; Delfia, Wallac, Finland) using two monoclonal antibodies directed against different sites of the 22-kDa variant of hGH. Intraassay and interassay coefficients of variation were less than 5 and 10%, respectively.

Genomic DNA isolation and PCR detection of mDlk1 plasmid
Total genomic DNA was isolated from different tissues using DNeasy isolation kit from Qiagen (via VWR, Albertslund, Denmark) according to the instructions of the manufacturer. The injected pUC-UBI-mDlk1 plasmid was detected by PCR using primers specific for mDlk1 cDNA as follows: mDlk1 forward, 5'-GCGTGG ACCTGGAGAAAG-3'; and mDlk1 reverse, 5'-GGAAGTCACCCCCGATGT-3'. PCR conditions were 95 C for 30 sec, 60 C for 30 sec, and extension for 72 C for 1 min for 35 cycles with 2 U Ampliqon tempase mastermix (Bie & Bernsten, Sandbaekvej, Denmark).

Phenotype analysis: total RNA isolation and real-time PCR analysis
Total RNA was isolated from freeze-dried mouse abdominal fat samples using single step method with TRIzol (Invitrogen, Tastrup, Denmark) according to the instructions of the manufacturer. The integrity and purity of total RNA was verified spectrophotometrically and by gel electrophoresis on 0.8% SeaKem agarose (BMA, Hellerup, Denmark). cDNA was synthesized from 5 µg total RNA using a commercial revertAid H minus first-strand cDNA synthesis kit (Fermentas, Copenhagen, Denmark) according to manual instructions. Real-time PCR was performed with the iCycler IQ detection system (Bio-Rad, Herlev, Denmark) by using SYBR Green I as a double-strand DNA-specific binding dye. Thermocycling was performed in a final volume of 20 µl containing 3 µl cDNA sample (diluted 1:20), 20 pmol of each primer, and 2x iQ SYBR Green Supermix (Bio-Rad). The quantification of each target gene and ß-actin mRNA was performed in separate tubes. Gene expression levels for each target gene were calculated using the comparative threshold cycle (CT) method [(1/(2{Delta}CT) formula, where {Delta}CT is the difference between CT target and CT reference] after normalization to ß-actin mRNA (User Bulletin no. 2 from PerkinElmer, Wellesley, MA). Data were analyzed using optical system software version 3.1 (Bio-Rad) and Microsoft (Seattle, WA) Excel 2000 to generate relative expression values.

Bone mass and body composition measurements
Total body mass (grams), fat mass (milligrams), bone mineral content (BMC) (milligrams), and bone mineral density (BMD) (milligrams per square centimeter) were measured using dual-energy x-ray absorptiometry (DEXA) by PIXImus Instrument (version 1.44; Lunar, Copenhagen, Denmark).

Microcomputer tomography (micro-CT) scanning
The fifth lumbar vertebra, right tibiae, and femurs of mice were carefully dissected and cleaned of soft tissues and kept at –20 C before micro-CT scanning. Three bone samples from each mouse were scanned with a high-resolution micro-CT system (µCT 40; Scanco Medical, Bassersdorf, Switzerland), resulting in three-dimensional (3-D) reconstruction of cubic voxel sizes 12 x 12 x 12 µm3. For the fifth lumbar vertebra, the entire vertebra was scanned, consisting of 150–250 micro-CT slide images, and 0.5 mm beneath both end plates was separated and used for analysis of cancellous and cortical bone tissues. For distal femur, each 3-D image dataset consisted of approximately 100 micro-CT slide images, and 50 slice images (600 µm) were used for analysis of subchondral bone tissues. For proximal tibia, each 3-D image dataset consisted of 100 micro-CT slide images, and 60 slice images (720 µm) were used for analysis of subchondral bone tissues (1024 x 1024 pixels) with 16-bit gray levels.

The microstructural properties of cancellous bone were calculated based on true, unbiased, and assumption-free 3-D methods. Bone volume (BV) fraction was computed based on the voxel size and the number of segmented voxels in the 3-D image, i.e. bone voxel per total specimen voxel (37). Structure model index (SMI) (a measure of predominant shapes in the structure, i.e. plate like, rod like, or a combination of plate and rod) was based on a differential analysis of the triangulated bone surface (BS) of a structure. The SMI value is zero for an ideal flat plate structure, and the SMI value is 3 for an ideal cylindrical rod structure (38). Trabecular thickness in millimeters and BS density in mm–1 were calculated as described previously (38). Unbiased and assumption-free quantification of connectivity was based on a topological approach, and connectivity density is the number of trabeculae per volume in mm–3. Meaningful microarchitectural parameters of cortical bone were quantified. These parameters were cortical thickness, determined in 3-D datasets (micrometer), BV fraction (percentage), BS density (mm–1), and mean pore size (micrometer). The cross-sectional area (square millimeters) of cortex was the mean value obtained from subchondral cortical bone. A detailed description for the quantification of 3-D microarchitecture of cortical bone has been presented previously (40).

The statistical analysis was performed using SPSS version 10.0.7 (SPSS, Chicago, IL). Linearity and equal variance of the data were checked. Two-sample t tests were used to compare difference in properties between the two groups. A P value < 0.05 was considered significant.

Bone histomorphometry
For bone dynamic histomorphometry, FA1 mice and controls were injected with calcein (20 mg/kg; Fluka Chemie, Buchs, Switzerland) and Alizarin red (20 mg/kg; Merck, Darmstadt, Germany) 8 and 3 d, respectively, before necropsy. Immediately after scarifying the animals, spine, femurs, and tibiae, were removed from each animal and placed in 70% ethanol until plastic embedding.

Tibiae were embedded in methylmetacrylate (Technovit 9100; Heraeus Kulzer, Wehrheim/Ts., Germany) and cut into 5- to 8-µm-thick sections. Fluorochrome-based dynamic histomorphometric measurements of bone formation were determined by an Axiophot photomicroscope (Zeiss, Oberkochen, Germany) linked to a camera (CF 15/4 MC; Kappa, Gleichen, Germany), and a QUANTIMET 600 image analysis system was used to calculate the amount of single-labeled surface per BS (percentage), double-labeled surface per BS (percentage), mineralized surface (MS)/BS (percentage), corrected mineral apposition rate (micrometers per day), BFR/BS (micrometers per day), and double-labeled BFR/BS (micrometers per day). The calculations of the dynamic parameters were performed as recommended previously (41).

Peripheral quantitative computed tomography (pQCT) densitometer
Measurements were made in the mid-diaphysis of the left tibia on a tomograph (XCT-960; Stratec, Pforzheim, Germany). A slice thickness of 1.2 mm and the smallest possible voxel size of 0.197 x 0.197 x 1 mm were chosen. Periosteal and endocortical perimeter of the cortical compartment were measured (42).

Statistical analysis
All values are expressed as mean ± SD or mean ± SEM. Comparison between groups was performed using unpaired Student’s t test (two-tailed). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of mice expressing high levels of serum FA1
To investigate the systemic effect of circulating FA1 on bone in vivo, we used HGTP to generate mice expressing high serum levels of FA1. For this purpose, we used the PHD184 expression plasmid that has been successfully used to give high and prolonged expression of hGH in mice for more than 6 months after only a single injection (33). In our preliminary experiments, we observed that injecting a pUC-UBI-mDlk1 construct led to elevation of serum FA1 within 2 d up to 15-fold compared with saline-injected control (198 ± 74.3 vs. 13.4 ± 1.1 ng/ml; P < 0.0005). These experiments confirmed the efficient expression of Dlk1 using this technique and the normal processing and shedding of membrane-bound dlk1 protein to produce the circulating soluble ectodomain FA1. We also observed that serum levels of FA1 started to decline after 10 d after injection (data not shown). To obtain persistent high serum levels of FA1, HGTP was repeated every 2 wk over a period of 2 months. A total of 16 mice were injected with pUC-UBI-mDlk1 plasmid and 11 mice with saline as control. The serum levels of FA1 were determined by ELISA 1 wk after each injection. Eleven mice with variable, but high serum FA1 (ranging from 44.18 ± 12.1 to 288.66 ± 39.5 ng/ml) were selected for phenotype analysis (Fig. 1CGo). Analysis of genomic DNA obtained from different tissues of FA1 mice revealed the presence of the injected plasmid in the liver only (Fig. 1AGo). This was further confirmed by immunohistochemical staining for mouse FA1 protein (Fig. 1BGo). Also, we did not detect any expression of the mFA plasmid in fat tissue or cultured bone cells from FA1 mice (data not shown). We detected no histological evidence of tissue damage in a variety of tissues (e.g. liver, heart, and lung) as a result of HGTP or the transgene expression (data not shown).


Figure 1
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  		FIG. 1. Generating mice with high serum levels of mdlk1/FA1 by HGTP. A, PCR analysis for tracking the injected pUC-UBI-mDlk1 plasmid. Genomic DNA was extracted from different tissues of FA1 mice, and PCR was performed using primers designed to amplify the pUC-UBI-mDlk1 construct. The transgene was only detected in the liver. B, Immunohistochemical staining for the tissue distribution of mdlk1/FA1 transgene. Liver sections obtained from FA1 mice were stained positive for mdlk1/FA1 protein (indicated by arrows) expressed by the injected plasmids. Tissues obtained from other organs were negative for the expression of the transgene, including fat tissue and cultured bone cells (data not shown). C, ELISA measurements of mFA1 in sera obtained from FA1 mice and controls (saline injected). Serum samples were collected 1 wk after each injection over a 2-month period. Values are represented as mean ± SD of four serum samples per mouse.

 
Phenotypic analysis of the mice
No differences were observed in physical activity levels between the FA1 mice and control mice, and high serum FA1 levels were not associated with excess morbidity or mortality. FA1 mice displayed significantly lowered body weight compared with their control group (33.5 ± 1.1 vs. 35.8 ± 0.1 g; P < 0.001) (Fig. 2AGo), whereas longitudinal bone length was similar in both FA1 mice and control groups as revealed by measuring of tibia length (FA1 mice, 18.40 ± 0.16 mm vs. control, 18.47 ± 0.10 mm; not significant).


Figure 2
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  		FIG. 2. Reduced body weight and total fat mass in mFA1 mice. A, Control mice (white bars) and FA1 mice (gray bars) were weighted gravimetrically on an XL-300 balance just 2 months after the first injection and before scarifying. B, Total fat mass was determined by densitometry using PIXImus2 imager (Lunar) and plotted against the corresponding serum level of mFA1 per each individual mFA1 mouse. C, Real-time PCR analysis of adipogenic expression markers in sc fat. Expression of each target gene was normalized to ß-actin. Values are represented as mean ± SD; n = 8–11 mice. *, P < 0.05 and **, P < 0.005 vs. control mice. APM1, Adiponectin; FAS, fatty acid synthase; PPAR{gamma}2, peroxisome proliferator-activated receptor-{gamma}2.

 
Reduced fat mass in FA1 mice
Analysis of body composition using DEXA scanning revealed a statistically significant reduction of total fat mass in FA1 mice compared with the control mice (3.6 ± 0.06 g vs. control, 3.8 ± 0.04 g; P < 0.01), and fat mass was inversely correlated with the FA1 serum levels in FA1-overexpressing mice (Fig. 2BGo). Moreover, we found that the steady-state levels of mRNA expression of several adipocytic differentiation markers, peroxisome proliferator-activated receptor-{gamma}2, adiponectin, fatty acid synthase, and adipsin were reduced by 50, 65, 37, and 75%, respectively, in the abdominal fat of FA1 mice (Fig. 2CGo).

Reduced bone mass in FA1 mice
To determine the effect of circulating FA1 on the bone phenotype, we first analyzed changes in total body BMC and BMD as well as BMC and BMD of femurs, tibiae, and spine using DEXA scanning. FA1 mice with serum mFA1 greater than 100 ng/ml displayed significantly decreased total BMC and BMD compared with controls (Fig. 3AGo). Interestingly, both total BMC and total BMD were inversely correlated with the levels of circulating FA1 in FA1 mice (Fig. 3Go, A and B).


Figure 3
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  		FIG. 3. Reduced total BMC and BMD in FA1 mice. Total BMC (grams) (A) and BMD (grams per square centimeter) (B) were measured using a PIXImus2 (Lunar) for control mice (white bars) and FA1 mice (gray bars). Values are represented as mean ± SD (n = 8–11) and also plotted vs. the corresponding serum mFA1 per each individual mFA1 mouse. **, P < 0.005 vs. control mice.

 
Marked inhibitory effects of FA1 on trabecular bone parameters
Detailed analysis of trabecular and cortical bone using micro-CT was performed in L5 vertebra, proximal tibia, and distal femur. The FA1 mice showed a 44 and 24% decrease in the trabecular BS per bone total volume (TV) and BV/TV, respectively, at distal femur (Fig. 4AGo). The same parameters were also significantly reduced when measured at the proximal tibia of FA1 mice (Fig. 4AGo). Serum levels of FA1 showed a significant negative correlation with the BV/TV in FA1 mice (Fig. 4BGo). In addition, micro-CT analysis of bone structural parameters at distal femur and proximal tibia of FA1 mice (Fig. 4CGo) revealed marked impairment of the trabecular bone microarchitecture with the presence of thinner (15% decrease) trabeculae and 27% decrease in connectivity. We also found lowered number of bone trabeculae (4.5 ± 0.8 vs. 5.8 ± 1.1 1/mm; P < 0.03) in FA1 mice compared with control mice (Fig. 4AGo). The changes in bone microarchitecture were associated with a trend toward a more rod-like trabecular structure in the FA1 mice as shown by 26% higher SMI than the control mice. Conversely, we found no significant differences in any measured cortical bone parameters at L5 vertebra, proximal tibia, and distal femur between FA1 mice and controls as measured by micro-CT (Table 1Go). To gain more detailed analysis on cortical bone, we also measured the cortical bone parameters at the mid-diaphysis of the tibia using pQCT. As indicated in Table 2Go, FA1 mice displayed significantly smaller periosteal and endocortical circumference but normal cortical BMD and cortical thickness (Table 2Go). Together, these data indicate that FA1 mice had smaller bone diameters but no changes in cortical thickness.


Figure 4
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  		FIG. 4. Decreased trabecular microarchitectural parameters in FA1 mice. A, 3-D micro-CT analysis was performed at the distal femurs and proximal tibia of the mice. All scanning had the same voxels size of 12 µm3 in the Scanco Medical µ-CT 40 as described in Material and Methods. Values of microarchitectural parameters of trabecular bone were measured and are represented as mean ± SD (n = 8; FA1 > 100 ng/ml). *, P < 0.05 vs. control mice. B, Inverse correlation between BV fractions of the distal femurs in FA1 mice and the corresponding serum mFA1. C, 3-D reconstruction of distal femur and proximal tibia from micro-CT images with median values for the FA1 mice and the control group. Tb/Th, Trabecular thickness; Tb Sp, trabecular space; TbN, trabecular number; CD, connectivity.

 

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  		TABLE 1. Cortical bone parameters measured by 3-D micro-CT

 

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  		TABLE 2. Cortical bone parameters measured by pQCT

 
Histomorphometric analysis of bone
To determine the cellular basis of decreased bone mass in FA1 mice, we performed dynamic histomorphometric analysis in the diaphysis endocortical surface of tibia. As shown in Fig. 5Go, the FA1 mice exhibited 39% decrease in MS/BS (P < 0.001) compared with the control group. Also, mineral apposition rate was reduced in FA1 mice (0.14 ± 0.1 vs. 0.34 ± 0.19 µm/d; P ≤ 0.05) as well as BFR (0.035 ± 0.02 vs. 0.13 ± 0.03; P < 0.02) compared with the control group (Fig. 5Go).


Figure 5
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  		FIG. 5. BFR is markedly reduced in FA1 mice. Dynamic histomorphometric analysis was performed in tibia mid-diaphysis endocortical surface after fluorescent imaging microscopy. MS/BS, mineral apposition rate (MAR), and BFR were significantly inhibited in FA1 mice. *, P < 0.05 and **, P < 0.005 vs. control mice.

 
Circulating FA1 acts downstream of the GH/IGF-I axis, and the serum level of FA1 is negatively modulated by GH
It has been shown previously that FA1 is colocalized with GH in somatotroph cells of the adult anterior pituitary gland (43), and its serum level is modulated in response to pharmacological changes in GH levels (44). Because GH is a major hormone participating in several physiologic processes, including bone and fat mass regulation, we studied the possible hormonal regulation of circulating FA1 with GH. For this purpose, the FA1 mice model and other mice models with either GH deficiency or GH overexpression generated by using HGTP (33) were used.

We first studied the influence of increased serum FA1 levels (in FA1 mice) on the serum levels of IGF-I and IGFBPs, including IGFBP-3, by RIA and Western ligand blot assays. As shown in Fig. 6Go, high levels of circulating FA1 did not result in significant changes in the basal serum levels of IGF-I or any of the IGFBPs. Also, there were no differences in the expression pattern of IGFI/IGFBPs in bone samples between FA1 mice and control group as assessed by real-time PCR analysis (data not shown). We then determined serum FA1 levels in mice expressing high serum GH levels. For this purpose, we used hGHhigh mice (GH, 1052.8 ± 416.3 ng/ml; n = 10) or hGHdiff mice (GH, 6–250 ng/ml; n = 24). Increased serum levels of GH significantly led to decreased serum levels of FA1 by 60% (from 14 ± 1.1 to 5.4 ± 0.09 ng/ml) in hGHhigh mice (Fig. 7AGo). In addition, GH was shown to reduce serum FA1 levels in a dose-dependent manner in hGHdiff mice, with an inverse correlation between serum GH levels and FA1 levels (Fig. 7BGo). To further study the relationship between serum levels of GH and FA1, we measured serum FA1 in HX mice that received either green fluorescent protein (GFP) plasmid as control (HX+GFP mice; n = 7) or GH expression construct (HX+GH mice) in HGTP (31, 36). Basal serum FA1 levels were markedly increased by 450% in HX mice compared with normal nonhypophysectomized mice (15 ± 2.8 vs. 70 ± 6.4 ng/ml, respectively; P < 0.001) (Fig. 7DGo). Furthermore, the GH therapy of HX mice using HGTP was efficient to normalize both the serum IGF-I levels to their normal levels (Fig. 7CGo) as well as reducing the increased serum FA1 by 40% in a dose-dependent manner (Fig. 7Go, D and E).


Figure 6
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  		FIG. 6. Increasing serum FA1 is not associated with changes in the serum pattern of IGF-I/IGFBPs. Levels of IGF-I and IGFBP-1–IGFBP-5 were measured in the sera collected from FA1 mice (gray bars; n = 11) and controls (white bars; n = 10). A, Serum IGF-I was measured by RIA. IGFBP-3 (B) and IGFBP-1, IGFBP-2, IGFBP-4, and IGFBP-5 (C) were measured from Western ligand blotting (WLB).

 

Figure 7
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  		FIG. 7. Modulation of serum mFA1 by changing the GH level under pathophysiological conditions. A, Male BALB/cA mice were hydrodynamically injected with a high dose of pUC-UBI-hGH plasmid (n = 10) to generate hGHhigh mice (GH, 1052.8 ± 416.3) or injected with saline solution as a control (n = 6). B, Male BALB/cA mice were hydrodynamically injected with different doses of hGH plasmid (hGHdiff mice; n = 24) to study the effect of hGH in a dose-dependent manner. C, Mice were hypophysectomized at 5 wk of age and hydrodynamically injected at 8 wk of age with either GFP plasmid (pEGFP-N1) (gray bars, HX+GHP; n = 7) or pUC-UBI-hGH plasmid (black bars, HX+GH; n = 9). Normal mice (nonhypophysectomized) without treatment were used as control (white bars; n = 7) as described previously (31 ). IGF-I and mFA1 measurements were performed as described in Materials and Methods. *, P < 0.05 and **, P < 0.005 vs. control mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we identified soluble FA1 as a novel endocrine regulator of bone and fat mass and demonstrate that some developmentally active molecules can acquire new functions in the adult organism. Increasing serum FA1 levels in mice led to decreased total body weight, total fat mass, and BMD in a dose-dependent manner, and the effects on bone were pronounced in the trabecular bone compartment. We also demonstrated that FA1 serum levels are inversely correlated with changes in serum GH levels under physiological and pathological conditions.

We used HGTP to study the long-term systemic effect of FA1 on bone and fat mass in adult animals. This method has been developed for nonviral gene therapy (for review, see Ref. 45) and has been used previously by our group successfully (33). The advantages of this procedure include simple methodology and the ability for testing the effect of a transgene in adult organisms, thus avoiding the confounding effects of development-related effects induced by the transgenes. In addition, the procedure generates a sufficient number of animals with variable serum levels of the transgene, allowing for dose-response studies. Our data demonstrated successful usage of HGTP to elevate serum levels of FA1. We also found that the main source of transgenic FA1 was the liver, in which the transgene was integrated and expressed without causing any tissue damage. In a previous study from our group, one single injection of pUC-UBI-GH plasmid was sufficient to obtain high and prolonged expression of GH in either immune-deficient or normal mice (33). However, the elevated FA1 levels using the same backbone plasmid declined 10 d after injection, and repeated HGTP every 2 wk was required to maintain high and stable levels of FA1. The differences between these experiments could be explained by the presence of anti-mFA1 antibodies. However, we were not able to detect antibodies formed against mFA1 in the FA1 mice (data not shown). Also, in GH experiments, the presence of antibodies against hGH did not affect its expression (33). Alternatively, the decreased level of mFA1 may be the result of feedback inhibition of the translation or shedding of FA1 by factors elicited by high levels of FA1. The nature of these factors remains to be determined.

Serum FA1 levels in our FA1 mice (198 ± 74.3 ng/ml) were biologically relevant because they lie within the physiological range of FA1 levels present during the life cycle of an organism and also in some pathological conditions. Serum FA1 levels in normal adult mice are 11.3 ± 5.0 ng/ml and in adult humans were 25.8 ng/ml (median age, 16–60 yr). Higher levels are seen in amniotic fluid (human and mouse, 25 µg/ml) in maternal serum during late pregnancy (in mice, ~400 ng/ml) or in sera of newborn mice (median, 15 µg/ml), and the later is more than 100 times higher than the levels obtained in FA1 mice (26, 27). Also, serum levels of FA1 are elevated in some human pathological conditions, e.g. neurofibromatosis (five times higher than the normal range) (46), renal failure (10 times higher) (26), and small cell lung cancer patients (10–1000 times higher) (47).

The high serum levels of FA1 in FA1 mice were biologically active because they led to the expected effects on fat mass. dlk1/FA1 has been demonstrated by a number of groups as an inhibitor of adipocyte differentiation in vitro (48, 49). The association of high levels of serum FA1 and decreased fat mass in vivo has also been observed in a number of studies. Transgenic mice overexpressing the soluble form of dlk1/Pref-1 under the aP2 or albumin promoter displayed reduced adipose tissue mass as a result of impaired adipocyte differentiation with reduced levels of adipocyteic hormones, leptin, and adiponectin (15). The expression of Dlk1/FA1 mRNA in epididymal fat was also upregulated in association with loss of fat in leptin treated wild-type Zucker diabetic fatty ZDF rats (51) and in the lipodystrophic transgenic mice overexpressing sterol-regulated element-binding protein 1c in adipose tissue (52). Conversely, increased fat mass was reported in Dlk1/FA1-deficient mice and in both humans and mice with the syndrome of maternal uniparental disomy (mUPD) (in which Dlk1/FA1 is silent; mUPD12 in mice, mUPD14 in humans) (16, 17, 53, 54). In addition to low fat mass, we observed that gene expression of adipocyte differentiation markers was impaired in FA1 mice compared with controls. These findings corroborate previous findings from Lee et al. (15) and suggest that decreased fat mass in the presence of high levels of FA1 may partially be attributable to impairment of adipocyte differentiation.

Increased levels of circulating FA1 was associated with decreased bone mass, and these effects were more pronounced in the trabecular compared with cortical compartment. The effects of bone mass were not attributable to changes in body size, which was comparable in FA1 and control mice. In a previous study using mice overexpressing the soluble form of Dlk1/FA1 under the aP2 promoter, the authors noted skeletal abnormalities, including short thoracic cavity with short ribs and fused vertebra in transgenic embryos and kinky-shaped tail in adult mice (15). However, no detailed analysis of the bone phenotype was reported. These skeletal malformations are most probably attributable to the effects of Dlk1/FA1 during bone development and not attributable to changes in the postnatal bone turnover. The experimental design used in the present experiments avoids this important confounder and suggests that FA1 may have direct effects on the skeletal turnover of adult animals. Bone histomorphometry analyses suggested that high levels of FA1 are inhibitory to osteoblastic activity and their recruitment or activation. This is consistent with our previously published data that demonstrated the ability of soluble FA1 to reversibly inhibit the late-stage differentiation of hMSC into osteoblasts in vitro and to inhibit newly formed bone in an ex vivo calvaria organ culture (14). Because it is unknown whether dlk/FA1 acts as a receptor or a ligand to modulate this differentiation signal (55), it is still unclear whether the effects of circulating FA1 on bone mass are the result of its interaction with osteoblasts or through other indirect mechanisms. In this context, we have shown recently that dlk1/FA1 can regulate the differentiation of hMSC, indirectly via mediating increased production of proinflammatory cytokines and other immune-response-related factors by hMSC (56). As a member of Notch-epidermal growth factor-like family, dlk1/FA1 was shown to be implicated in the interaction between hematopoietic stem cells and stromal cells in bone marrow microenvironment that mediate both self-renewal and differentiation of hematopoietic stem cells (55, 57, 58). Therefore, it is plausible that FA1 exerts its effects on bone phenotype of FA1 mice via altering the hematopoietic-derived cells and their microenvironment in the bone marrow. These hypotheses need to be confirmed.

Decreased fat mass and body weight is known to have deleterious effects on bone mass, as shown in patients with rapid weight loss (59) and in anorexia nervosa (60). It is unlikely that the observed effects on bone mass in FA1 mice are secondary to changes in body weight or fat mass because these effects occurred simultaneously with changes in body weight. Also, the above mentioned direct effects of FA1 on osteoblastic cells suggest direct effects on bone.

Dlk1/FA1 has been recognized as a developmental molecule with defined effects during development. However, the expression of FA1 by hormonal producing cells in several adult endocrine tissues suggested physiological functions for dlk1/FA1 in postnatal organisms (55). Our findings demonstrate that the biological function of dlk1/FA1 extends beyond development and plays a role in the adult organism in regulating bone and fat homeostasis, and the presence of a hormonal regulatory mechanism controlling serum levels of FA1 support this hypothesis. We identified GH as a possible regulator of FA1 serum levels. A relationship between GH and dlk1/FA1 has been recognized previously as a result of the coexpression of FA1 and GH by somatotroph cells in the adult anterior pituitary gland (43). We found an inverse relationship between serum GH and serum FA1 levels. It does not seem that FA1 exerts a regulatory role on GH secretion per se because no changes in the GH/IGF system was detected in FA1 mice with high levels of FA1.

GH may exert its modulatory effects on dlk1/FA1 at several levels. GH can directly or indirectly regulate the transcription, translation, proteolysis/shedding, and/or clearance of FA1. In support of the direct effect of GH on dlk1/FA1, GH was shown to stimulates the steady-state mRNA expression levels of Dlk1 and the processing of its soluble form FA1 in cultured pancreatic ß-cells in vitro (18, 61). Also, the antiadipogenic effects of GH on cultured primary rat adipocytes is associated with sustained induction of Dlk1/FA1 expression (39). Recently, ADAM17/TACE has been identified as a putative protease mediating the cleavage and the shedding of dlk1 ectodomain (25). Interestingly, TACE is also responsible for the GH receptor as a result of cleavage and the generating of circulating GH binding protein (50), suggesting that similar biological situations are associated with changes in serum levels of GH and FA1. It is plausible that the GH can regulate the processing of dlk1/FA1 by affecting its ectodomain shedding by TACE. Also, because GH exerts major biological effects on bone and fat, it is plausible that changes in FA1 may mediate part of the biological effects of GH. However, these questions need additional investigation.


   Acknowledgments
 
We thank Mrs. Andrea Venturiere-Rebmann (Novartis Pharma AG) for assistance with bone histomorphometry. Tina K. Nielsen, Anette Kliem, Inga Bisgaard, Karen Mathiassen, and Kirsten Nyborg are thanked for excellent technical assistance.


   Footnotes
 
This work was supported by Danish Medical Research Council, Danish Center for Stem Cell Research, the Karen Elise Jensen Foundation, and the Novo Nordisk Foundation.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online April 19, 2007

Abbreviations: BFR, Bone formation rate; BMC, bone mineral content; BMD, bone mineral density; BS, bone surface; BV, bone volume; CT, threshold cycle; DEXA, dual-energy x-ray absorptiometry; 3-D, three-dimensional; dlk1, delta-like 1; FA1, fetal antigen 1; GFP, green fluorescent protein; h, human; hGHhigh, mice with high serum levels of hGH; hGHdiff, mice with different serum levels of hGH; HGTP, hydrodynamic gene transfer procedure; HX, hyphysectomized; IGFBP, IGF binding protein; m, mouse; micro-CT, microcomputer tomography; MS, mineralized surface; MSC, mesenchymal stem cell; mUPD, maternal uniparental disomy; pQCT, peripheral quantitative computed tomography; pref-1, preadipocyte factor 1; SMI, structural model index; TV, total volume.

Received February 5, 2007.

Accepted for publication April 9, 2007.


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 Materials and Methods
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