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Endocrine Research Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. B. Lawrence Riggs, Mayo Clinic, 200 First Street SW, North 6 Plummer, Rochester, Minnesota 55905. E-mail address: riggs.lawrence{at}mayo.edu
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2,
transcription factors involved in commitment to the osteoblast and
adipocyte pathways, respectively. Thus, leptin acts on human marrow
stromal cells to enhance osteoblast differentiation and to inhibit
adipocyte differentiation. Our data support the hypothesis that leptin
is a previously unrecognized, physiological regulator of these two
differentiation pathways, acting primarily on maturation of stromal
cells into both lineages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several clinical studies have demonstrated that body fat and bone mass are directly related (14, 15, 16, 17). Although mechanical loading may contribute to this relationship, the direct relationship remains regardless of whether the skeletal site is weight bearing (18), suggesting that other factors are also involved. Increased conversion of androgens to estrogens by peripheral aromatization in adipose tissue has been thought to be one possible causal mechanism (19). In addition, we have considered the possibility that leptin may be the hormonal mediator relating fat mass and bone mass. Serum leptin levels are increased in obesity and correlate positively with fat mass (20). This observation led us to investigate the action of leptin on osteoblastic differentiation and function in vitro.
In this study, we evaluated the effects of recombinant human leptin on the conditionally immortalized human marrow stromal cell line, hMS2–12, with the potential to differentiate to either osteoblastic or adipocytic lineages (21). Our data show that leptin enhances osteoblastic differentiation of marrow progenitors and inhibits late adipocytic differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin D3, [3H]thymidine,
and [-32P]deoxy (d)-CTP were obtained from
DuPont New England Nuclear (Boston, MA).
L-Ascorbic acid phosphate was purchased from Wako Pure Chemical Industries Ltd. (Richmond, VA). Kits for the
measurement of osteocalcin and procollagen protein were gifts from
Metra Biosystem (Mountain View, CA). Leptin was provided by Eli Lilly & Co. (Indianapolis, IN).
Cell culture
The conditionally immortalized human marrow stromal (hMS) cell
lines were established in our laboratory by transfecting the hMS cells
with a gene coding for a temperature-sensitive mutant (tsA58) of simian
virus 40 large T antigen (SV40LTA) (21). As previously reported (21),
incubation of the cells at 34 C, the permissive temperature for
SV40LTA, increases the rate of cell proliferation and inhibits
differentiation until confluence. At 39.5 C, the restrictive
temperature, SV40LTA is consistently inactive, little cell division
occurs, and the cells begin to differentiate. Because the six cell
lines that we characterized displayed a homogeneous phenotype (21), we
used the hMS2–12 cell line for these studies.
hMS2–12 cells were maintained in a humidified atmosphere at 34 C in
5% CO2 in MEM containing 10% (vol/vol)
heat-inactivated FBS (HI-FBS), geneticin (G418; 0.2 µg/ml), and 1%
stock penicillin (10,000 U/ml)-streptomycin (10,000 µg/ml), hereafter
termed standard growth medium. Medium was changed twice a week. To
assess the effects of leptin on the shunting between adipocytic and
osteoblastic lineages, the study required culture conditions in which
the hMS cell lines have an equal propensity to differentiate toward
either osteoblasts or adipocytes. Thus, as previously demonstrated
(Gori, F., et al., manuscript submitted for publication),
all experiments were performed in a medium (hereafter termed standard
differentiation medium) containing 10% HI-FBS, 10-8
M dexamethasone (DEX), 10-8 M
1,25-dihydroxyvitamin D3, 10 mM
ß-glycerolphosphate, and 100 µM L-ascorbate
phosphate in the presence of freshly prepared leptin or vehicle
(phosphate buffer disodium, 120 µM final concentration;
pH 7.5), unless otherwise indicated.
The preadipocyte cell line 3T3-L1 was used as a control for the
expression of peroxisome proliferator-activated
receptor-2 (PPAR2). Cells were obtained
from American Type Culture Collection (Manassas, VA) and
maintained in MEM, with nonessential amino acids and Earle’s
Balanced Salt Solution, and 10% calf serum.
Western immunoblot for leptin receptor
Western blot analysis for leptin receptor (OB-R) was performed
using a rabbit polyclonal IgG epitope affinity-purified anti-OB-R
antibody against the common form of OB-R (ABR, Golden, CO).
Cells were plated at a density of 2 x 104 cells/cm2 in T75 flasks in standard growth medium and maintained for 4 days at 34 C. They were then washed twice in PBS and cultured in standard differentiation medium at 39.5 C, in the presence of 0.6 µg/ml leptin or vehicle. After 6 days, cells were washed twice with PBS, and the pellet was suspended in electrophoresis buffer and electrophoresed in a 7.5% SDS-PAGE under reducing conditions, using a protein mixture (Amersham, Arlington Heights, IL) as standards. The blots were then electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). A hematopoietic cell line, K562, was used as a positive control.
The blots were blocked for 2 h in Tris-HCl phosphate buffer (TBS; pH 7.4) containing 0.1% (vol/vol) Tween-20 and 0.1% (wt/vol) BSA (blocking buffer), and then hybridized in blocking buffer with an anti OB-R antibody (1 µg/ml) at 4 C. After overnight incubation the blots were washed twice with PBS containing 0.1% (vol/vol) Tween-20 and incubated in blocking buffer for 2 h with a peroxidase-conjugated affinity pure IgG goat antirabbit (1:10,000 final dilution). After three washes in TBS containing 0.1% (vol/vol) Tween-20, immunoreactive proteins were visualized using the ECL chemiluminescence detection kit (Amersham) according to the manufacturer’s instructions.
Assessment of cell proliferation
Cell proliferation was assessed by [3H]thymidine
incorporation. Cells were plated at a density of 2 x
104 cells/well in 24-well microtiter plates in standard
growth medium. After 48 h at 34 C, cells were washed twice in PBS
and incubated at 34 C for an additional 24 h in serum-free MEM
and 0.1% (wt/vol) BSA to synchronize the cell population. Cells were
then incubated in standard differentiation medium in the presence of
leptin (0.6 µg/ml) or vehicle for 48 h at 34 or 39.5 C. To
assess DNA synthesis, 1 µCi [3H]thymidine was added for
the last 24 h of incubation. Cells were harvested by
trypsinization, and [3H]thymidine was extracted by
trichloroacetic precipitation and detected by scintillation
counting.
Semiquantitative RT-PCR
Cells were plated at a density of 1.8 x 105
cells/well in six-well microtiter plates in standard growth medium and
maintained for 4 days at 34 C. They were then washed twice in PBS and
cultured for various time intervals in standard differentiation medium
at 39.5 C in the presence of leptin (0.15–2.4 µg/ml) or vehicle.
Total cellular RNA was extracted using the RNA STAT-60 kit following
the manufacturer’s instructions. Complementary DNA (cDNA) was
synthesized from 2 µg total RNA in a 20-µl reaction mix containing
4 µl of 5 x incubation buffer for AMV reverse transcriptase; 50
pmol poly(deoxythymidine)15 primer; 20 nmol each of dATP,
dCTP, dGTP, and dTTP; 20 U ribonuclease inhibitor; and 20 U AMV reverse
transcriptase. The reaction time was 1 h at 42 C.
Aliquots of 1 µl cDNA were amplified in a 25-µl PCR mixture that
contained 2.5 µl of 10 x Expand high fidelity PCR buffer with
15 mM MgCl2; 5 pmol 5'- and 3'-oligo primers;
2.5 nmol each of dATP, dCTP, dGTP, and dTTP; 0.25 µl
[-32P]dCTP (10 µCi/µl); and 0.35 U Expand high
fidelity Taq DNA polymerase. Each cDNA sample was amplified
in duplicate PCR for each gene. Amplification reactions were performed
in a GeneAmp 9600 thermal cycler (Perkin Elmer, Norwalk,
CT), for the following cDNAs: adipsin, bone/liver/kidney alkaline
phosphatase (AP), type I collagen (Col I), core-binding factor-a1
(Cbfa1), leptin, lipoprotein lipase (LPL), common region of OB-R
variants, long form of OB-R, osteocalcin (OC), and
PPAR2. The housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was amplified as a control for RNA loading and
variations in cDNA synthesis efficiency. After initial determination of
the linear phase of amplification, reactions were performed for 28–35
cycles depending on product intensity, except for GAPDH, which was
performed for 24 cycles. All PCR reactions were conducted by annealing
at 55 C and ended in a 7-min incubation at 72 C. Primer sequences for
these genes have been reported previously (21), except for adipsin,
leptin, PPAR2, and OB-R. A 251-bp cDNA fragment of
adipsin (sense, 5'-GGTCACCCAAGCAACAAAGT-3'; antisense,
5'-CCTCCTGCGTTCAAG-TCATC-3'), a 227-bp cDNA fragment of leptin (sense,
5'-GCTTTGGCCCTATCTTT-TCT-3'; antisense, 5'-CACGTTTCTGGAAGCAAC-3'), and
a 390-bp cDNA fragment of PPAR2 (sense,
5'-CAGTGGGGATG-CTCATAA-3'; antisense, 5'-CTTTTGGCAT-ACTCTGTGAT-3') were
amplified for 30–35 cycles with denaturation at 94 C for 30 min,
annealing at 55 C for 30 min, and extension at 72 C for 30 min. A
375-bp fragment from a region common to all OB-R variants (sense,
5'-TGTTGTGAATGTCTTGTGCC-3'; antisense, 5'-TACTCCAGTCACTCCAGATTCC-3')
and a 240-bp fragment specific to the long form variant of the OB-R
(sense, 5'-ATAGTTCAGTCACCAAGTGC-3'; antisense,
5'-GTCCTGGAGAACT-CTGATGTCC-3') were amplified, using the same
conditions. Cbfa1 primers were as reported by Komori et al.
for amplification of the mouse Cbfa1 gene (22); they amplified a 267-bp
fragment starting at nucleotide 136 of the human cDNA sequence with
98% homology between the amplified fragment and human sequences.
PCR products were analyzed as described previously (21). Briefly, 9-µl samples were electrophoresed on a 1.5% (wt/vol) agarose gel containing 0.01% (wt/vol) ethidium bromide. Visualized PCR product bands were excised from the gel, and radioactivity within gel slices was quantitated using a Beckman Coulter, Inc. LS600 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Quantification of PCR product was normalized to the GAPDH PCR product. The cDNA from three separate RNA samples were analyzed for each gene and condition. The different gene products were purified using the Wizard PCR Preps DNA kit. For sequence analysis, approximately 150 ng of each purified cDNA fragment were added to 3.2 pmol of either 5'- or 3'-primer and analyzed in both directions in an automated DNA sequence analyzer.
Assays of bone-related proteins
AP activity. Cells were plated at a density of 2 x
104 cells/well in 48-well microtiter plates in standard
growth medium and allowed to adhere for 4 days at 34 C. They were
washed twice in PBS and further incubated in standard growth medium at
39.5 C in the presence of leptin (0.6 µg/ml) or vehicle for 3, 6, and
9 days or in the presence of increasing doses of leptin (0.075–2.4
µg/ml). AP enzyme activity was quantitated in cell lysate by
spectrophotometric measurement of p-nitrophenol release at
37 C (23).
Measurement of Col I and OC proteins. Cells were plated at a
density of 8 x 104 cells/well in 12-well microtiter
plates in standard growth medium and allowed to proliferate for 4 days
at 34 C. Cells were then washed twice in PBS and incubated at 39.5 C in
standard differentiation medium in the presence of leptin (0.6 µg/ml)
or vehicle for 21 days. Medium was changed every 3 days and replaced in
all conditions 24 h before harvest with 1 ml MEM containing
0.1% (wt/vol) BSA. Conditioned medium was collected from 12–21 days
of culture and measured for Col I (Prolagen-C, Metra Biosystem,
Mountain View, CA) and OC (Novocalcin, Metra Biosystem) proteins by
enzyme-linked immunosorbent assay. Results were then normalized to
total cellular protein values, as measured in cell lysates by the
Bradford method (Bio-Rad Laboratories, Inc., Hercules,
CA).
Assay of mineralized matrix formation
Cells were plated at a density of 8 x 104
cells/well in 12-well microtiter plates in standard growth medium and
allowed to proliferate for 4 days at 34 C. They were then washed twice
in PBS and incubated at 39.5 C in standard differentiation medium in
the presence of leptin (0.6 µg/ml) or vehicle for 21 days. Medium was
changed every 3 days and replaced in all conditions 24 h before
harvest with 1 ml MEM containing 0.1% (wt/vol) BSA. After
collecting conditioned medium, the extent of mineralized matrix was
determined by Alizarin Red S staining. Briefly, cells were fixed in
70% ethanol for 1 h at room temperature, then washed with PBS and
stained with 40 mM Alizarin Red S, pH 4.2, for 10 min at
room temperature. Next, cell preparations were washed five times with
deionized water and incubated in PBS for 15 min to eliminate
nonspecific staining. The stained matrix was assessed using a
Nikon Diaphot inverted microscope and was photographed
using a Nikon 35-mm camera (Nikon, Tokyo,
Japan). As described by Bodine et al. (24), Alizarin Red S
staining was released from cell matrix by incubation in
cetyl-pyridinium chloride for 15 min. The amount of released dye was
quantified by spectrophotometry at 540 nm. Results were then normalized
to total cellular protein values, as measured in cell lysate by the
Bradford method (Bio-Rad Laboratories, Inc.).
Assessment of cytoplasmic lipid droplet formation
Cells were plated at a density of 5 x 104
cells/well in 12-well microtiter plates in standard growth medium and
allowed to proliferate for 4 days at 34 C. Cells were then washed in
PBS and incubated in standard differentiation medium at 39.5 C in the
presence of leptin (0.6 µg/ml) or vehicle for 6, 9, 12, and 15 days.
Cytoplasmic inclusions of neutral lipids were assessed by Oil Red O
staining. The percentage of Oil Red O-positive cells was determined by
counting cells in 30 contiguous fields/well after random starts.
PPAR2 expression by Western blot
analysis
To evaluate the protein expression of PPAR2 in
the hMS2–12 cell line, Western blot analysis was performed using a
rabbit polyclonal IgG epitope affinity-purified
anti-PPAR2 antibody (ABR). Cells were plated at a
density of 2 x 104 cells/cm2 in T75
flasks in standard growth medium and maintained for 4 days at 34 C.
They were then washed twice in PBS and cultured in standard
differentiation medium at 39.5 C in the presence of leptin (0.6
µg/ml) or vehicle for 1 and 3 days. The 3T3-L1 preadipocyte cell line
was grown to confluence and then cultured either in its standard medium
or in the presence of insulin (1 µM),
3-isobutyl-1-methylxanthine (200 µM), and DEX
(10-6 M; 3T3-L1 differentiation medium) for 6
days. Cells were washed twice with PBS, the pellets were suspended in
electrophoresis buffer, and equal aliquots of total cell lysates (50
µg total protein) were electrophoresed in 7.5% SDS-PAGE under
reducing conditions, using a protein mixture (Amersham) as standards.
The blots were then electrotransferred onto a nitrocellulose membrane
(Schleicher & Schuell, Inc.). They were blocked for 2
h in TBS (pH 7.4) containing 0.1% (vol/vol) Tween-20 and 5% (vol/vol)
milk (blocking buffer) and then hybridized in blocking buffer with an
anti PPAR2 antibody (1 µg/ml) at 4 C. After overnight
incubation, the blots were washed twice with TBS containing 0.1%
(vol/vol) Tween-20 and incubated in blocking buffer for 2 h with a
peroxidase-conjugated affinity-purified IgG goat antirabbit (1:10,000
final dilution). Blotting with the secondary antibody alone was
performed as a control for the specificity of the reagent. After three
washes in TBS containing 0.1% (vol/vol) Tween-20, immunoreactive
proteins were visualized using the ECL chemiluminescence detection kit
(Amersham) according to the manufacturer’s
instructions.
Statistical analysis
All values are expressed as the mean ± SEM.
Two-sample Student’s t test was used to evaluate
differences between the stimulated sample and the respective control.
Multiple measurement ANOVA was used for dose- and time-dependent
differences. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
gene expression of the different OB-R variants was increased in cells
cultured at 39.5 C, when the cells stop proliferating and
differentiate, as assessed by semiquantitative RT-PCR (data not shown).
By Western blot analysis (Fig. 1B), the short form of OB-R (
120 kDa)
was present in hMS2–12 cells at both 34 and 39.5 C, whereas the long
form of OB-R (230 kDa) was present only at 39.5 C. We further
evaluated gene expression of the long form OB-R in the hMS2–12 cells
that were cultured for 6 days under either purely adipogenic conditions
(i.e. MEM containing 15% rabbit serum, 10-8
M DEX, 10-8 M
1,25-dihydroxyvitamin D3, 200 µM
isobutylmethylxanthine, and 50 µg/ml insulin) or purely osteogenic
conditions (i.e. MEM containing 10% HI-FBS,
10-8 M DEX, 10-8 M
1,25-dihydroxyvitamin D3, 10 mM
ß-glycerolphosphate, and 100 µM L-ascorbate
phosphate), as previously described (21). No difference was observed
between these conditions as assessed by semiquantitative RT-PCR. A
trend in decreased gene expression was induced under leptin treatment
as the adipogenic characteristic of the medium increased (data not
shown).
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).
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). However, after 3 days at 39.5 C, leptin dose
dependently increased gene expression of early osteoblastic
differentiation markers, AP and Col I, by maximums of 66%
(P < 0.03) and 145% (P < 0.001),
respectively, compared with that in vehicle culture. The level of OC
mRNA began to increase at 3 days and increased up to 147% by 6 days in
a dose-dependent manner (P < 0.001; Fig. 3).
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, leptin significantly increased AP
activity of hMS2–12 cells in a time (P = 0.02)- and
dose (P = 0.002)-dependent manner, by 24% after 3 days
in culture at 39.5 C and by 42% after 9 days, compared with the
control value. Production of type I procollagen and OC proteins also
increased significantly with time, by 72% (P < 0.01)
and 37% (P < 0.01), respectively. Maximal osteocalcin
production coincided with the onset of matrix mineralization (Fig. 5).
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).
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). Interestingly, gene expression
of LPL, an early marker of adipocytic differentiation, was increased by
leptin in a dose-dependent manner (P < 0.001) by 3
days (Fig. 7) and remained higher than control thereafter.
PPAR2 gene expression was nonsignificantly increased
over the interval of 30 min to 72 h after leptin exposure (Fig. 8
). No further changes were observed up
to 9 days of treatment (data not shown).
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).
Whereas stained lipid droplets consistently appeared after 6 days at
39.5 C in both groups, leptin treatment progressively reduced lipid
accumulation compared with the control value beginning after 9 days of
leptin treatment (P = 0.02).
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2 protein production2 protein
content in cell lysates after leptin administration by Western blot
analysis. Immunoblotting showed a 58-kDa band, consistent with the
expected mol wt of PPAR2. Leptin did not change
PPAR2 protein levels after 1 and 3 days (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We showed that the hMS2–12 cells were targets for leptin action by demonstrating that they expressed mRNA and protein for the leptin receptor. The short form of the receptor was present at both the permissive (34 C) and the restrictive (39.5 C) temperature, whereas the long splice variant was present in significant quantities only at the restrictive temperature. Gene expression for the different variants of OB-R increased when the cells were cultured at 39.5 C regardless of the culture conditions (i.e. adipogenic, osteogenic, or both). Thus, alteration in OB-R expression appears to be induced by the occurrence of reduced proliferation and increased differentiation rather than the particular differentiation pathway. However, little is known about the regulation of long form OB-R expression and its specific activity. In fact, its presence has been reported in both early and lineage-restricted hematopoietic progenitors (12), in the placenta and in different fetal tissues (28), and in mature brown and white adipose tissues (29). Kellerer et al. could not detect the long form of OB-R in C2C12 myotubes, but leptin was able to activate Janus kinase-2- and insulin receptor substrate-2-dependent pathways in these cells (30). Only fasting has been recently reported to be associated with increased expression of mRNA for the long form of the OB-R in the hypothalamus (31). In our study, leptin administration had a modest inhibiting effect on the gene expression of its own receptor only in adipogenic conditions.
We were unable to demonstrate a significant effect of leptin on proliferation of hMS2–12 cells at either temperature. Leptin has been shown to exert a proliferative effect on hematopoietic progenitors (12, 13) and on pancreatic cells (32), but this does not appear to be the case for osteoblastic precursor cells. We cannot rule out that the absence or presence in low concentration of the long form of the OB-R might have contributed to this lack of proliferative effect at 34 C, as shown in the BaF3 hematopoietic cell line (33).
In contrast to the lack of an effect on proliferation, leptin clearly exerted a dose-dependent increase on osteoblast differentiation. These effects appear to be at the level of maturation rather than at the level of commitment. Cbfa1 is a recently discovered early response gene that is involved in commitment to the osteoblast differentiation pathway (22, 25, 26). We were unable to identify either early (hours) or late (days) effects of leptin on Cbfa1 gene expression. In contrast, there were consistent dose-dependent effects on steady state levels of mRNA and protein production of the osteoblast maturation markers AP, OC, and type I procollagen. Moreover, leptin treatment increased the mineralization of matrix, the hallmark of the osteoblast phenotype. The mechanism by which leptin increases osteoblastic differentiation is unclear. However, OB-R is closely related to the gp130 protein (34), and leptin binding to OB-R stimulates phosphorylation of the Jak/STAT kinase cascade, as do other gp130-dependent inducers of osteoblastic gene transcription, such as oncostatin M and leukemia inhibitory factor (35, 36).
The effects on adipocyte differentiation were more complex.
PPAR2 is an early response gene that is involved in
commitment to the adipocyte pathway (37). We failed to find a
significant effect of leptin administration on mRNA expression or
protein production of PPAR2. Steady state mRNA levels
for LPL, a gene expressed early in the adipocyte differentiation
pathway (38), were increased, whereas those for adipsin and leptin,
genes that are expressed later during differentiation, were decreased.
Most importantly, the accumulation of cytoplasmic lipid droplets, the
hallmark of the adipocyte phenotype, was decreased by leptin,
indicating that the overall effect of leptin was to decrease adipocyte
differentiation. This decrease in neutral lipid accumulation is
consistent with the findings of earlier studies showing that leptin
lowers lipogenesis in the preadipocytic cell line 30A5 (10) and
triglyceride accumulation in transfected rat pancreatic islets
(11).
The reason for the paradoxical increase in expression of LPL despite the presence of an overall decrease in adipocyte differentiation is unclear. Interestingly, leptin administration to rodents increased LPL and decreased leptin gene expression in adipose tissue (29, 39). Also, overexpression of LPL in transgenic mice did not lead to an increase in fat deposition, but, rather, caused a decrease in the plasma triglyceride level (40). Thus, it is possible that induction of LPL may provide energy for marrow stromal cell differentiation by enhancing the hydrolysis of extracellular triglycerides for cells early in either the adipocyte or osteoblast pathway.
The effects of leptin on marrow stromal cell osteoblastic differentiation differ from those of bone morphogenetic protein-2 in this model system (Gori, F., et al; manuscript submitted for publication). First, the effect of leptin on osteoblast differentiation was less pronounced quantitatively. Second, the primary effect of bone morphogenetic protein-2 appears to be on commitment to the osteoblast pathway through an early increase in Cbfa1 gene expression, whereas leptin did not affect Cbfal expression. Thus, leptin appears to primarily act at the level of osteoblast maturation, rather than at the level of commitment. Because a recent study found that human bone marrow adipocytes in primary culture had high leptin expression (41), leptin could serve as an autocrine/paracrine factor to modulate the differentiation of marrow stromal cells as well as hematopoietic precursor cells (12, 13) in addition to its well established endocrine role.
Our observations may be relevant to the clinical observations that obesity is associated with increased bone mineral density (14, 42, 43, 44) and that increased body mass index protects against postmenopausal bone loss (45, 46). These associations have generally been attributed to the mechanical effect of increased load bearing on increasing bone formation and to the effect of higher circulating estrogen levels associated with increased aromatase activity in the larger mass of adipose tissue (19). However, fat mass and bone mineral density (BMD) are still directly and strongly correlated after adjusting for differences in serum estrogen levels (47, 48). Moreover, fat mass (49) or body weight (50) and BMD are correlated in women independent of menopausal status. As obesity is also associated with higher circulating leptin levels (20), it is possible that the effect of leptin on enhanced maturation of marrow precursor cells into osteoblasts is a major factor in mediating the relationship between fat mass and BMD. Indeed, a recent clinical study demonstrated a direct relationship between serum leptin level and total body bone area in pubertal girls (51).
In summary, we report here a direct osteogenic effect of leptin on a human marrow stromal cell line with the capability to differentiate to either osteoblasts or adipocytes. We found that leptin enhances osteoblastic differentiation and inhibits adipocytic differentiation. Leptin appears to act by enhancing the entire osteoblast maturation pathway and by inhibiting the late adipocytic maturation pathway, rather than by acting at the level of commitment to either pathway. Thus, leptin could serve as a previously unrecognized physiological regulator of the balance between the fat and bone compartments. The possibility that leptin could be useful as a therapeutic agent for the treatment of osteoporosis deserves evaluation.
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Acknowledgments |
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Footnotes |
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Received June 22, 1998.
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A. Martin, V. David, L. Malaval, M.-H. Lafage-Proust, L. Vico, and T. Thomas Opposite Effects of Leptin on Bone Metabolism: A Dose-Dependent Balance Related to Energy Intake and Insulin-Like Growth Factor-I Pathway Endocrinology, July 1, 2007; 148(7): 3419 - 3425. [Abstract] [Full Text] [PDF]
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 T. Hajri, A. M. Hall, D. R. Jensen, T. A. Pietka, V. A. Drover, H. Tao, R. Eckel, and N. A. Abumrad CD36-Facilitated Fatty Acid Uptake Inhibits Leptin Production and Signaling in Adipose Tissue Diabetes, July 1, 2007; 56(7): 1872 - 1880. [Abstract] [Full Text] [PDF]
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 N. Pischon, N. Heng, J.-P. Bernimoulin, B.-M. Kleber, S.N. Willich, and T. Pischon Obesity, Inflammation, and Periodontal Disease Journal of Dental Research, May 1, 2007; 86(5): 400 - 409. [Abstract] [Full Text] [PDF]
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 J. B. Richards, A. M. Valdes, K. Burling, U. C. Perks, and T. D. Spector Serum Adiponectin and Bone Mineral Density in Women J. Clin. Endocrinol. Metab., April 1, 2007; 92(4): 1517 - 1523. [Abstract] [Full Text] [PDF]
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S. M. Turner, S. Roy, H. S. Sul, R. A. Neese, E. J. Murphy, W. Samandi, D. J. Roohk, and M. K. Hellerstein Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1101 - E1109. [Abstract] [Full Text] [PDF]
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 M. W. Hamrick Leptin and Bone: A Consensus Emerging? IBMS BoneKEy, March 1, 2007; 4(3): 99 - 107. [Abstract] [Full Text] [PDF]
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 A. M. Pino, J. M. Rodriguez, S. Rios, P. Astudillo, L. Leiva, G. Seitz, M. Fernandez, and J P. Rodriguez Aromatase activity of human mesenchymal stem cells is stimulated by early differentiation, vitamin D and leptin J. Endocrinol., December 1, 2006; 191(3): 715 - 725. [Abstract] [Full Text] [PDF]
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 R. C. Johnson, J. A. Leopold, and J. Loscalzo Vascular Calcification: Pathobiological Mechanisms and Clinical Implications Circ. Res., November 10, 2006; 99(10): 1044 - 1059. [Abstract] [Full Text] [PDF]
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E. M. Clark, A. R. Ness, J. H. Tobias, and and the Avon Longitudinal Study of Parents and Chi Adipose Tissue Stimulates Bone Growth in Prepubertal Children J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2534 - 2541. [Abstract] [Full Text] [PDF]
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 S. A. Shapses and C. S. Riedt Bone, Body Weight, and Weight Reduction: What Are the Concerns? J. Nutr., June 1, 2006; 136(6): 1453 - 1456. [Abstract] [Full Text] [PDF]
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 B. Wagoner, D. B. Hausman, and R. B. S. Harris Direct and indirect effects of leptin on preadipocyte proliferation and differentiation Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1557 - R1564. [Abstract] [Full Text] [PDF]
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 S Prouteau, L Benhamou, and D Courteix Relationships between serum leptin and bone markers during stable weight, weight reduction and weight regain in male and female judoists. Eur. J. Endocrinol., March 1, 2006; 154(3): 389 - 395. [Abstract] [Full Text] [PDF]
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 T. G. Ramsay Porcine preadipocyte proliferation and differentiation: A role for leptin? J Anim Sci, September 1, 2005; 83(9): 2066 - 2074. [Abstract] [Full Text] [PDF]
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A. Martin, R. de Vittoris, V. David, R. Moraes, M. Begeot, M.-H. Lafage-Proust, C. Alexandre, L. Vico, and T. Thomas Leptin Modulates both Resorption and Formation while Preventing Disuse-Induced Bone Loss in Tail-Suspended Female Rats Endocrinology, August 1, 2005; 146(8): 3652 - 3659. [Abstract] [Full Text] [PDF]
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 J H Davies, B A J Evans, and J W Gregory Bone mass acquisition in healthy children Arch. Dis. Child., April 1, 2005; 90(4): 373 - 378. [Abstract] [Full Text] [PDF]
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 B. A Watkins, Y. Li, H. E Lippman, S. Reinwald, and M. F Seifert A test of Ockham's razor: implications of conjugated linoleic acid in bone biology Am. J. Clinical Nutrition, June 1, 2004; 79(6): 1175S - 1185S. [Abstract] [Full Text] [PDF]
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C. M. Morberg, I. Tetens, E. Black, S. Toubro, T. I. A. Soerensen, O. Pedersen, and A. Astrup Leptin and Bone Mineral Density: A Cross-Sectional Study in Obese and Nonobese Men J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 5795 - 5800. [Abstract] [Full Text] [PDF]
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 A. Ghazali, F. Grados, R. Oprisiu, D. Bunea, P. Moriniere, N. El Esper, I. El Esper, M. Brazier, J. C. Souberbielle, A. Fournier, et al. Bone mineral density directly correlates with elevated serum leptin in haemodialysis patients Nephrol. Dial. Transplant., September 1, 2003; 18(9): 1882 - 1890. [Abstract] [Full Text] [PDF]
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G. S. Kim, J. S. Hong, S. W. Kim, J.-M. Koh, C. S. An, J.-Y. Choi, and S.-L. Cheng Leptin Induces Apoptosis via ERK/cPLA2/Cytochrome c Pathway in Human Bone Marrow Stromal Cells J. Biol. Chem., June 6, 2003; 278(24): 21920 - 21929. [Abstract] [Full Text] [PDF]
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J. N. Roemmich, P. A. Clark, C. S. Mantzoros, C. M. Gurgol, A. Weltman, and A. D. Rogol Relationship of Leptin to Bone Mineralization in Children and Adolescents J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 599 - 604. [Abstract] [Full Text] [PDF]
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S. Khosla Editorial: Leptin--Central or Peripheral to the Regulation of Bone Metabolism? Endocrinology, November 1, 2002; 143(11): 4161 - 4164. [Full Text] [PDF]
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M. Kveiborg, R. Chiusaroli, N. A. Sims, M. Wu, G. Sabatakos, W. C. Horne, and R. Baron The Increased Bone Mass in {Delta}FosB Transgenic Mice Is Independent of Circulating Leptin Levels Endocrinology, November 1, 2002; 143(11): 4304 - 4309. [Abstract] [Full Text] [PDF]
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E. F. Gevers, N. Loveridge, and I. C. A. F. Robinson Bone Marrow Adipocytes: A Neglected Target Tissue for Growth Hormone Endocrinology, October 1, 2002; 143(10): 4065 - 4073. [Abstract] [Full Text] [PDF]
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 F. Machinal-Quelin, M. N. Dieudonne, M. C. Leneveu, R. Pecquery, and Y. Giudicelli Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways Am J Physiol Cell Physiol, April 1, 2002; 282(4): C853 - C863. [Abstract] [Full Text] [PDF]
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H. Blain, A. Vuillemin, F. Guillemin, R. Durant, B. Hanesse, N. de Talance, B. Doucet, and C. Jeandel Serum Leptin Level Is a Predictor of Bone Mineral Density in Postmenopausal Women J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1030 - 1035. [Abstract] [Full Text] [PDF]
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 K. Kume, K. Satomura, S. Nishisho, E. Kitaoka, K. Yamanouchi, S. Tobiume, and M. Nagayama Potential Role of Leptin in Endochondral Ossification J. Histochem. Cytochem., February 1, 2002; 50(2): 159 - 170. [Abstract] [Full Text] [PDF]
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 M. Haberland, A. F. Schilling, J. M. Rueger, and M. Amling Brain and Bone: Central Regulation of Bone Mass : A New Paradigm in Skeletal Biology J. Bone Joint Surg. Am., December 1, 2001; 83(12): 1871 - 1876. [Full Text] [PDF]
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M. Sato, N. Takeda, H. Sarui, R. Takami, K. Takami, M. Hayashi, A. Sasaki, S. Kawachi, K. Yoshino, and K. Yasuda Association between Serum Leptin Concentrations and Bone Mineral Density, and Biochemical Markers of Bone Turnover in Adult Men J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5273 - 5276. [Abstract] [Full Text] [PDF]
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 B. A. Watkins, Y. Li, and M. F. Seifert Nutraceutical Fatty Acids as Biochemical and Molecular Modulators of Skeletal Biology J. Am. Coll. Nutr., October 1, 2001; 20(90005): 410S - 416. [Abstract] [Full Text]
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B. Burguera, L. C. Hofbauer, T. Thomas, F. Gori, G. L. Evans, S. Khosla, B. L. Riggs, and R. T. Turner Leptin Reduces Ovariectomy-Induced Bone Loss in Rats Endocrinology, August 1, 2001; 142(8): 3546 - 3553. [Abstract] [Full Text] [PDF]
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 J. J. Minguell, A. Erices, and P. Conget Mesenchymal Stem Cells Experimental Biology and Medicine, June 1, 2001; 226(6): 507 - 520. [Abstract] [Full Text] [PDF]
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J. A. Pasco, M. J. Henry, M. A. Kotowicz, G. R. Collier, M. J. Ball, A. M. Ugoni, and G. C. Nicholson Serum Leptin Levels Are Associated with Bone Mass in Nonobese Women J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 1884 - 1887. [Abstract] [Full Text]
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T. A. DRAKE, E. SCHADT, K. HANNANI, J. M. KABO, K. KRASS, V. COLINAYO, L. E. GREASER III, J. GOLDIN, and A. J. LUSIS Genetic loci determining bone density in mice with diet-induced atherosclerosis Physiol Genomics, April 27, 2001; 5(4): 205 - 215. [Abstract] [Full Text] [PDF]
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T. A Ricci, S. B Heymsfield, R. N Pierson Jr, T. Stahl, H. A Chowdhury, and S. A Shapses Moderate energy restriction increases bone resorption in obese postmenopausal women Am. J. Clinical Nutrition, February 1, 2001; 73(2): 347 - 352. [Abstract] [Full Text] [PDF]
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 G. Fantuzzi and R. Faggioni Leptin in the regulation of immunity, inflammation, and hematopoiesis J. Leukoc. Biol., October 1, 2000; 68(4): 437 - 446. [Abstract] [Full Text]
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O. Ogueh, S. Sooranna, K. H. Nicolaides, and M. R. Johnson The Relationship between Leptin Concentration and Bone Metabolism in the Human Fetus J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1997 - 1999. [Abstract] [Full Text]
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F. Parhami, Y. Tintut, A. Ballard, A. M. Fogelman, and L. L. Demer Leptin Enhances the Calcification of Vascular Cells : Artery Wall as a Target of Leptin Circ. Res., May 11, 2001; 88(9): 954 - 960. [Abstract] [Full Text] [PDF]
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