Abstract
Leptin, critical in regulation of energy metabolism, is also important for normal bone growth, maturation and turnover. Compared to wild type (WT) mice, bone mass is lower in leptin-deficient ob/ob mice. Osteopenia in growing ob/ob mice is due to decreased bone accrual, and is associated with reduced longitudinal bone growth, impaired cancellous bone maturation and increased marrow adipose tissue (MAT). However, leptin deficiency also results in gonadal dysfunction, disrupting production of gonadal hormones which regulate bone growth and turnover. The present study evaluated the role of increased estrogen in mediating the effects of leptin on bone in ob/ob mice. Three-month-old female ob/ob mice were randomized into one of the 3 groups: (1) ob/ob + vehicle (veh), (2) ob/ob + leptin (leptin) or (3) ob/ob + leptin and the potent estrogen receptor antagonist ICI 182,780 (leptin + ICI). Age-matched WT mice received vehicle. Leptin (40 µg/mouse, daily) and ICI (10 µg/mouse, 2×/week) were administered by subcutaneous injection for 1 month and bone analyzed by X-ray absorptiometry, microcomputed tomography and static and dynamic histomorphometry. Uterine weight did not differ between ob/ob mice and ob/ob mice receiving leptin + ICI, indicating that ICI successfully blocked the uterine response to leptin-induced increases in estrogen levels. Compared to leptin-treated ob/ob mice, ob/ob mice receiving leptin + ICI had lower uterine weight; did not differ in weight loss, MAT or bone formation rate; and had higher longitudinal bone growth rate and cancellous bone volume fraction. We conclude that increased estrogen signaling following leptin treatment is dispensable for the positive actions of leptin on bone and may attenuate leptin-induced bone growth.
Introduction
Leptin plays a role in regulating the rate of bone elongation, maturation of primary spongiosa, cortical and cancellous bone accrual, and cancellous bone turnover (Turner et al. 2013). Leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice have bone- and bone compartment-specific alterations in bone microarchitecture; reduced bone length, mass and density; and reduced bone quality (Williams et al. 2011, Jing et al. 2016). The growth plates of ob/ob mice are abnormal due, in part, to poorly organized collagen fibril arrangement (Kishida et al. 2005). The pathological manifestations in growth plate are associated with decreased type X collagen expression, increased chondrocyte apoptosis and premature mineralization (Kishida et al. 2005), suggesting that leptin modulates events associated with terminal differentiation of chondrocytes. Importantly, the defects in growth plate and bone microarchitecture are largely reversed in growing ob/ob mice following administration of leptin (Kume et al. 2002, Maor et al. 2002, Kishida et al. 2005).
Osteoblast and osteoclast differentiation and/or function are also impaired in leptin signaling-deficient ob/ob and db/db mice (Turner et al. 2013). The lower bone formation generally reported in these mice is primarily due to reduced osteoblast number, although reduced osteoblast activity has also been noted. The dramatic increase in bone marrow adipose tissue (MAT) in long bones of leptin signaling-deficient mice suggests that leptin regulates bone marrow mesenchymal stem cell lineage decision; in the absence of leptin there is a net increase in marrow adipocytes and a decrease in osteoblasts. In contrast to a reduction in osteoblast perimeter, osteoclast perimeter is either normal or increased in ob/ob and db/db mice (Turner et al. 2013). However, bone resorption is reduced, implicating impaired osteoclast activity. The osteoclast defect in these mice contributes to growth plate abnormalities and development of mild osteopetrosis: the pathological retention of calcified cartilage into adulthood in bone in ob/ob and db/db mice likely contributes to the reduced bone quality noted in these animals (Jing et al. 2016).
Although absence of leptin signaling is ultimately responsible for the skeletal abnormalities in ob/ob and db/db mice, leptin is a pleiotropic hormone, impacting many physiological systems, including energy partitioning, thermogenesis, immune regulation, and gonadal function, each of which could independently influence bone metabolism (Mantzoros et al. 2011). Leptin is required for the release of gonadotropin-releasing hormone (GnRH) from the pituitary and, as a consequence, female ob/ob mice have greatly reduced estrogen levels and exhibit low uterine weight (Barash et al. 1996). GnRH-dependent estrogen synthesis and release play an important role in regulating bone elongation, accrual and turnover, and growing estrogen-deficient rodents exhibit skeletal abnormalities, including accelerated bone growth, increased bone turnover and cancellous bone loss (Turner et al. 1994, Burguera et al. 2001).
As mentioned, administration of leptin to ob/ob mice reverses many, if not all, of the skeletal abnormalities associated with leptin deficiency (Hamrick et al. 2005, Kishida et al. 2005, Iwaniec et al. 2007, Bartell et al. 2011). However, leptin administration to ob/ob mice also restores gonadal function (Barash et al. 1996) and in WT mice accelerates the onset of puberty (Ahima et al. 1997). Thus, it is possible that estrogen deficiency contributes to the skeletal phenotype observed in leptin signaling-deficient rodents and restoration of estrogen receptor signaling following leptin administration may influence the physiological response of ob/ob mice to leptin. This possibility is consistent with the results of a recent leptin dose response study conducted in ob/ob mice (Philbrick et al. 2017). Specifically, a pronounced dose-dependent stimulatory effect of leptin on bone accrual plateaued at a dose rate that increased uterine weight (an index of estrogen levels). Therefore, the current study was designed to determine the precise role of estrogen signaling in the skeletal response to leptin. This was accomplished by administering leptin sc to ob/ob mice in the presence or absence of the potent estrogen receptor antagonist ICI 182,780 (ICI) (Wakeling & Bowler 1992). Treatment with ICI replicates the uterine and skeletal responses to ovariectomy (ovx) in rodents, and thus its administration to leptin-treated ob/ob mice should prevent actions mediated through increased estrogen levels (Sibonga et al. 1998).
Materials and methods
Experimental design
One-month-old ob/ob and WT mice were purchased from Jackson Laboratory and maintained until 3 months of age. The ob/ob mice were then randomized by weight into one of 3 treatment groups: (1) ob/ob + vehicle (veh) (n = 5), (2) ob/ob + leptin (leptin) (n = 8) or (3) ob/ob + leptin + ICI (leptin + ICI) (n = 8). A group of WT mice (WT) (n = 9) received vehicle. Recombinant mouse leptin (CYT-351, Prospec Bio, Rehovot, Israel) was administered once daily by subcutaneous (s.c.) injection at a dose of 40 µg/mouse/day in 0.1 mL phosphate buffered saline (vehicle). ICI was administered 2×/week by sc injection at a dose of 10 µg/mouse in 0.1 mL extra virgin olive oil. Fluorochromes were administered at 9 days (declomycin, 15 mg/kg; Sigma Chemical), 4 days (calcein, 15 mg/kg; Sigma Chemical) and 1 day (calcein) prior to necropsy to label mineralizing bone. Food (Teklad 8604, Harlen Laboratories, Indianapolis, IN, USA) and water were provided ad libitum to all animals. Body weight was recorded daily. Food intake was also recorded daily, except for days 12–14, 19–20 and 26–28 when it was averaged over a 2–3 day interval. The mice were maintained at room temperature (22°C) and singly housed on a 12 h light–12 h darkness cycle for the duration of study. For tissue collection, mice were anesthetized using isoflurane and killed by cardiac exsanguination followed by decapitation. Uteri and abdominal white adipose tissue (WAT) were excised and weighed. Femora and 5th lumbar vertebrae were removed, fixed for 24h in 10% buffered formalin and stored in 70% ethanol for dual energy absorptiometry (DXA), microcomputed tomography (µCT) and histomorphometric analyses. The experimental protocol was approved by the Institutional Animal Care and Use Committee in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Densitometry
Total femur bone mineral content (BMC, g), bone area (cm2) and bone mineral density (BMD, g/cm2) were measured using DXA (Piximus 2, Lunar Corporation, Madison, WI, USA).
Microcomputed tomography
Microcomputed tomography was used for nondestructive 3-dimensional evaluation of bone volume and architecture. Femora and lumbar vertebrae were scanned in 70% ethanol using a Scanco µCT40 scanner (Scanco Medical AG, Bassersdorf, Switzerland) at a voxel size of 12 x 12 x 12 µm (55 kVp X-ray voltage, 145 µA intensity and 200 ms integration time). Filtering parameters sigma and support were set to 0.8 and 1, respectively. Voxels, having a threshold ≥245 (0–1000), were used to distinguish bone from non-bone. Total femur mineralized tissue volume (cancellous + cortical bone) was evaluated. This was followed by site-specific evaluation of cortical bone in the midshaft femur and cancellous bone in the distal femur metaphysis and epiphysis. For the femoral midshaft, 20 slices (0.24 mm) of bone were evaluated and total cross-sectional tissue volume (cortical and marrow volume, mm3), cortical volume (mm3), marrow volume (mm3), cortical thickness (µm) and polar moment of inertia (mm4, an index of bone strength in torsion) were measured. For the femoral metaphysis, 42 slices (0.50 mm) of cancellous bone were measured 45 slices (0.54 mm) proximal to the growth plate boundary. For the femoral epiphysis, the entire cancellous compartment (33 ± 1 slices) between the distal epiphyseal growth plate and distal femoral cortical shell was measured. µCT analysis of lumbar vertebrae was performed on cancellous bone within the vertebral body, between the cranial and caudal growth plates. Irregular manual contouring a few voxels interior to the endocortical surface was used to delineate cancellous from cortical bone. Direct cancellous bone measurements in the distal femur metaphysis and epiphysis and in lumbar vertebra included cancellous bone volume fraction (bone volume/tissue volume, %), trabecular thickness (µm), trabecular number (/mm), and trabecular separation (µm).
Histomorphometry
Distal femora were dehydrated in a graded series of ethanol and xylene, and embedded undecalcified in modified methyl methacrylate as described (Iwaniec et al. 2008). Coronal sections (4 µm thick) were cut with a vertical bed microtome (Leica 2065) and affixed to gel coated slides. One section per animal was stained for tartrate resistant acid phosphatase and counter stained with toluidine blue and used for cell-based measurements. A second section was left unstained for dynamic histomorphometry. Histomorphometric data were collected with a 20× objective using the OsteoMeasure System (OsteoMetrics, Inc., Atlanta, GA, USA). The sampling site for the distal femoral metaphysis was located 0.25–1.25 mm proximal to the growth plate and 0.1 mm from cortical bone.
Cell-based measurements included osteoblast perimeter (osteoblast perimeter/bone perimeter, %), osteoclast perimeter (osteoclast perimeter/bone perimeter, %), marrow adiposity (adipocyte area/tissue area, %), adipocyte density (number of adipocytes/tissue area, #/mm2) and adipocyte size (µm2). Osteoblasts, osteoclasts and adipocytes were identified as previously described (Iwaniec et al. 2016). Fluorochrome-based measurements of bone formation included mineralizing perimeter (mineralizing perimeter/bone perimeter: cancellous bone perimeter covered with double plus half single label normalized to bone perimeter, %), mineral apposition rate (the mean distance between two fluorochrome markers that comprise each double label divided by the 3-day interlabel interval, µm/day), and bone formation rate adjusted for bone perimeter (bone formation rate/bone perimeter: calculated by multiplying mineralizing perimeter by mineral apposition rate normalized to bone perimeter, µm2/µm/year). In addition, longitudinal growth rate was determined as the mean distance from the declomycin label to the mineralizing growth plate cartilage divided by the 9-day interval from label administration to sacrifice. All bone histomorphometric data are reported using standard 2-dimensional nomenclature (Dempster et al. 2013).
Statistics
Longitudinal data on body weight and food intake were analyzed using multivariate linear regression models with separate linear (body weight) or constant (food intake) time trends across groups. Candidate covariance models included independence, compound symmetric with and without equal variance across time and group, autoregressive of order 1, and moving average of order 1. Model selection was based on the Bayesian information criterion.
The principal goal of the analyses of tissues harvested at necropsy was to determine whether combination treatment (leptin + ICI) differed from leptin treatment. To accomplish this goal, mean responses of individual variables were compared for WT, vehicle, leptin and leptin + ICI mice using one-way analysis of variance, with Dunnett’s used to adjust for making multiple comparisons to the leptin + ICI group. The Kruskal–Wallis nonparametric test was used when only the normality assumption was violated, and a modified F-test was used when the assumption of equal variance was violated (Welch 1951); in these cases, the Wilcoxon–Mann–Whitney test or Welch’s two-sample t-test was used for pairwise comparisons and the Holm procedure was used to adjust for multiple comparisons (Holm 1979). The required conditions for valid use of Gaussian analysis of variance models were assessed using Levene’s test for homogeneity of variance, plots of residuals versus fitted values, normal quantile plots, and the Anderson–Darling test of normality. Data analysis was performed using R version 3.3.2 (R Core Team 2015). Differences were considered significant at P ≤ 0.05. All data are expressed as mean ± s.e.
Results
The effects of leptin deficiency and treatment with leptin and leptin + ICI on body weight and food consumption in ob/ob mice are shown in Fig. 1A and B, respectively. Vehicle-treated ob/ob mice weighed more and consumed more food than WT mice throughout the 4-week study. A progressive decrease in weight was observed in leptin-treated mice but weight remained higher than in WT mice for the duration of treatment. Food consumption decreased in leptin-treated and leptin + ICI treated ob/ob mice during the initial week of treatment. Mice treated with leptin + ICI did not differ from leptin-treated mice in body weight or food intake.
Effects of 1 month of daily sc leptin injection (40 µg/mouse/day) in the presence or absence of the potent estrogen receptor antagonist ICI 182,780 (ICI) on body weight (A) and food intake (B) in female ob/ob mice. Data are mean ± s.e., n = 5–9/group. aDifferent from WT mice, P < 0.05. bDifferent from vehicle-treated ob/ob mice, P < 0.05.
Citation: Journal of Endocrinology 233, 3; 10.1530/JOE-17-0103
The effects of treatment on abdominal WAT weight and uterine weight in ob/ob mice are shown in Fig. 2A and B, respectively. WAT weight in leptin + ICI treated mice was higher than in WT mice, lower than in vehicle-treated mice but did not differ from leptin-treated mice. In contrast, uterine weight in leptin + ICI treated mice was lower than in WT mice, did not differ from vehicle-treated mice and was lower than in leptin-treated mice.
Effects of 1 month of daily sc leptin injection (40 µg/mouse/day) in the presence or absence of the potent estrogen receptor antagonist ICI 182,780 (ICI) on abdominal white adipose tissue (WAT) weight (A) and uterine weight (B) in female ob/ob mice. Data are mean ± s.e., n = 5–9/group. aLeptin + ICI treated ob/ob mice different from vehicle-treated WT mice, P < 0.05; bleptin + ICI treated ob/ob mice different from vehicle-treated ob/ob mice, P < 0.05; cleptin + ICI treated ob/ob mice different from leptin-treated ob/ob mice, P < 0.05.
Citation: Journal of Endocrinology 233, 3; 10.1530/JOE-17-0103
The effects of treatment on femur DXA measurements, and microcomputed tomography measurements of cortical bone architecture in the midshaft femur, and cancellous bone architecture in the distal femur metaphysis and epiphysis in ob/ob mice are shown in Table 1. Leptin + ICI treated mice did not differ from WT mice in total femur BMC, bone area, BMD or bone volume. Femur length and cortical thickness were lower in leptin + ICI treated mice compared to WT mice while cross-sectional bone volume, marrow volume and polar moment of inertia were higher. Bone volume fraction in distal femur metaphysis and epiphysis was higher and trabecular spacing lower in the leptin + ICI treated mice compared to WT mice and this was due to higher trabecular number and thickness in the metaphysis and higher trabecular number in the epiphysis. Leptin + ICI treated mice had longer femurs and higher BMC and BMD than vehicle-treated mice. Similarly, cortical volume, thickness and polar moment of inertia were higher in the leptin + ICI treated mice. Bone volume fraction did not differ between leptin + ICI treated and vehicle-treated mice in the metaphysis but was higher in the leptin + ICI treated mice in the epiphysis. The latter was associated with lower trabecular number and higher trabecular thickness in the leptin + ICI treated mice. Leptin + ICI treated mice did not differ from leptin-treated mice in femur length, femur bone volume or cortical bone architecture. However, the two groups differed in cancellous bone architecture; leptin + ICI treated mice had higher cancellous bone volume fraction in the metaphysis which was associated with higher trabecular number, no change in trabecular thickness and lower trabecular spacing.
Effects of 1 month of daily sc leptin injection (40 µg/mouse/day) in the presence or absence of the estrogen receptor antagonist ICI 182,780 (ICI) on femur bone mineral content and density and on cortical bone architecture in midshaft femur, and cancellous bone architecture in distal femur metaphysis and epiphysis in female ob/ob mice.
| End point | WT + Veh | ob/ob + Veh | ob/ob + Lep | ob/ob + Lep + ICI | ANOVA P value |
|---|---|---|---|---|---|
| Dual energy X-ray absorptiometry | |||||
| Total femur | |||||
| BMC (g) | 0.019 ± 0.000 | 0.016 ± 0.001 | 0.017 ± 0.000 | 0.019 ± 0.001b | 0.0011 |
| Bone area (cm²) | 0.40 ± 0.01 | 0.38 ± 0.01 | 0.40 ± 0.01 | 0.41 ± 0.01 | 0.1000 |
| BMD (g/cm2) | 0.047 ± 0.001 | 0.041 ± 0.001 | 0.044 ± 0.000 | 0.046 ± 0.001b | 0.0010 |
| Microcomputed tomography | |||||
| Total femur | |||||
| Length (mm) | 15.2 ± 0.1 | 13.5 ± 0.2 | 14.0 ± 0.1 | 14.0 ± 0.1ab | 0.0000 |
| Bone volume (mm3) | 16.2 ± 0.2 | 13.5 ± 0.5 | 15.1 ± 0.3 | 15.6 ± 0.3b | 0.0000 |
| Mishaft femur (cortical bone) | |||||
| Cross-sectional volume (mm3) | 0.34 ± 0.00 | 0.38 ± 0.00 | 0.38 ± 0.01 | 0.40 ± 0.01a | 0.0000 |
| Cortical volume (mm3) | 0.16 ± 0.00 | 0.14 ± 0.00 | 0.16 ± 0.00 | 0.16 ± 0.00b | 0.0002 |
| Marrow volume (mm3) | 0.19 ± 0.00 | 0.24 ± 0.01 | 0.23 ± 0.01 | 0.24 ± 0.01a | 0.0000 |
| Cortical thickness (µm) | 183 ± 3 | 151 ± 3 | 167 ± 3 | 164 ± 2ab | 0.0000 |
| Polar moment of inertia (mm4) | 0.29 ± 0.01 | 0.30 ± 0.01 | 0.34 ± 0.01 | 0.35 ± 0.01ab | 0.0004 |
| Distal femur metaphysis (cancellous bone) | |||||
| Bone volume/tissue volume (%) | 4.4 ± 0.4 | 6.6 ± 1.1 | 6.2 ± 0.5 | 7.9 ± 0.3ac | 0.0003 |
| Trabecular number (/mm) | 3.8 ± 0.1 | 4.0 ± 0.2 | 3.8 ± 0.1 | 4.1 ± 0.04ac | 0.0046 |
| Trabecular thickness (μm) | 40 ± 1 | 42 ± 2 | 44 ± 1 | 43 ± 1a | 0.0431 |
| Trabecular spacing (μm) | 273 ± 5 | 261 ± 15 | 275 ± 4 | 257 ± 4ac | 0.0293 |
| Distal femur epiphysis (cancellous bone) | |||||
| Bone volume/tissue volume (%) | 25.9 ± 0.5 | 26.5 ± 0.3 | 29.9 ± 0.8 | 31.4 ± 0.6ab | 0.0001 |
| Trabecular number (/mm) | 5.1 ± 0.2 | 7.4 ± 0.4 | 6.3 ± 0.2 | 6.6 ± 0.2ab | 0.0000 |
| Trabecular thickness (μm) | 61 ± 1 | 50 ± 0 | 59 ± 1 | 60 ± 1b | 0.0000 |
| Trabecular spacing (μm) | 200 ± 5 | 136 ± 4 | 156 ± 4 | 148 ± 4.2a | 0.0000 |
Data are mean ± s.e., n = 5–9/group.
Leptin + ICI treated ob/ob mice different from vehicle-treated WT mice, P ≤ 0.05; bleptin + ICI treated ob/ob mice different from vehicle-treated ob/ob mice, P ≤ 0.05; cleptin + ICI treated ob/ob mice different from leptin-treated ob/ob mice, P ≤ 0.05.
The effects of treatment on cancellous bone microarchitecture in the 5th lumbar vertebra of ob/ob mice are shown in Table 2. Leptin + ICI treated mice had higher bone volume fraction than WT mice; the higher cancellous bone volume fraction was associated with higher trabecular number, no difference in trabecular thickness and lower trabecular spacing. Leptin + ICI treated mice had lower bone volume fraction than vehicle-treated mice. However, significant differences were not detected between the two groups in any of the architectural endpoints evaluated. Bone volume fraction, connectivity density, trabecular number and trabecular spacing did not differ between leptin + ICI and leptin-treated mice.
Effects of 1 month of daily sc leptin injection (40 µg/mouse/day) in the presence or absence of the estrogen receptor antagonist ICI 182,780 (ICI) on cancellous bone architecture in lumbar vertebra in female ob/ob mice.
| End point | WT + Veh | ob/ob + Veh | ob/ob + Lep | ob/ob + Lep + ICI | ANOVA P value |
|---|---|---|---|---|---|
| Bone volume/tissue volume (%) | 14.5 ± 0.5 | 24.4 ± 1.2 | 20.9 ± 0.9 | 20.1 ± 1.4ab | 0.0000 |
| Trabecular number (/mm) | 4.0 ± 0.1 | 5.6 ± 0.1 | 5.2 ± 0.2 | 5.4 ± 0.1a | 0.0000 |
| Trabecular thickness (μm) | 43 ± 0 | 44 ± 1 | 44 ± 1 | 43 ± 1 | 0.4650 |
| Trabecular spacing (μm) | 255 ± 8 | 175 ± 5 | 189 ± 6 | 184 ± 3.4a | 0.0000 |
Data are mean ± s.e., n = 5–9/group.
Leptin + ICI treated ob/ob mice different from vehicle-treated WT mice, P ≤ 0.05; bleptin + ICI treated ob/ob mice different from vehicle-treated ob/ob mice, P ≤ 0.05.
The effects of treatment on dynamic bone histomorphometry in distal femur metaphysis of ob/ob mice are shown in Fig. 3A, B, C and D. Leptin + ICI treated mice had higher longitudinal growth rate and mineralizing perimeter than WT mice but significant differences between the two groups were not detected in mineral apposition rate or bone formation rate. Leptin + ICI treated mice had higher values for each of these endpoints than vehicle-treated mice. Leptin + ICI treated mice had higher longitudinal growth rate than leptin-treated mice but mineralizing bone perimeter, mineral apposition rate and bone formation rate did not differ between the two groups.
Effects of 1 month of daily sc leptin injection (40 µg/mouse/day) in the presence or absence of the potent estrogen receptor antagonist ICI 182,780 (ICI) on longitudinal bone growth rate (A), mineralizing perimeter (B), mineral apposition rate (C), bone formation rate (D), osteoblast perimeter (E), osteoclast perimeter (F), marrow adiposity (G), adipocyte density (H) and adipocyte size (I) in distal femur metaphysis in female ob/ob mice. Representative photomicrographs illustrating differences in fluorochrome labeling in WT mice treated with vehicle (J), ob/ob mice treated with vehicle (K), ob/ob mice treated with leptin (L) and ob/ob mice treated leptin and ICI (M). Data are mean ± s.e., n = 5–9/group. aLeptin + ICI treated ob/ob mice different from vehicle-treated WT mice, P < 0.05; bleptin + ICI treated ob/ob mice different from vehicle-treated ob/ob mice, P < 0.05; cleptin + ICI treated ob/ob mice different from leptin-treated ob/ob mice, P < 0.05. Scale bar, 50 µm.
Citation: Journal of Endocrinology 233, 3; 10.1530/JOE-17-0103
The effects of treatment on osteoblast perimeter, osteoclast perimeter and MAT in the distal femur metaphysis of ob/ob mice are shown in Fig. 3E, F, G, H and I. Leptin + ICI treated mice had higher osteoblast perimeter, MAT area, adipocyte density and adipocyte size than WT mice but osteoclast perimeter did not differ between the two groups. Leptin + ICI treated mice had higher osteoblast perimeter and lower osteoclast perimeter, MAT area and adipocyte density than vehicle-treated mice. Leptin + ICI and leptin-treated mice did not differ in any of the cellular endpoints evaluated. The differences among groups in fluorochrome label and MAT can be readily appreciated in Fig. 3J, K, L and M.
Discussion
Oestrogens regulate bone growth and turnover and are essential for the sexual dimorphism of the skeleton (Turner et al. 1994). In growing rodents, endogenous estrogens promote termination of longitudinal bone growth, and have bone- and bone compartment-specific effects on bone acquisition and turnover balance (Turner et al. 1992, 1993). ICI is a fluorinated steroid, which binds to estrogen receptors with high affinity and lowers estrogen receptor levels by enhancing their proteosomal degradation (Wakeling & Bowler 1992, Dauvois et al. 1993). When administered to growing, ovary-intact rodents, ICI, similar to ovx, results in uterine atrophy, increased longitudinal bone growth and cancellous bone loss (Sibonga et al. 1998).
Leptin increases longitudinal bone growth in ob/ob mice (Kishida et al. 2005). The further increase in growth rate in ob/ob mice treated with leptin + ICI provides evidence that the concurrent increase in estrogen levels during leptin treatment antagonizes the growth promoting actions of leptin (Turner et al. 1994). Cancellous bone volume fraction was also higher in the distal femur metaphysis of ICI treated ob/ob mice compared to leptin-treated ob/ob mice. This finding was not necessarily anticipated because estrogen deficiency typically results in decreased cancellous bone volume fraction due to reduced trabecular number. However, this positive skeletal effect of ICI on cancellous bone volume fraction was associated with the noted accelerated longitudinal bone growth. Concurrent increases in longitudinal growth and bone formation rates are a plausible explanation for the higher cancellous bone volume fraction in leptin + ICI treated mice. This interpretation is consistent with prior studies demonstrating that sc administration of leptin reduces bone loss in growing ovx rats by attenuating the decrease in trabecular number (Burguera et al. 2001).
Ovx results in a reduction in cortical thickness in mice that is preventable by estrogen replacement (Hawse et al. 2014). In the present study, cortical thickness was increased in hypogonadal ob/ob mice following leptin treatment when compared to untreated ob/ob mice but cortical thickness was not restored to WT levels. Prior studies reported reduced endocortical bone formation in WT mice following adoptive transfer of bone marrow from leptin receptor-deficient db/db mice, and increased endocortical bone formation in ob/ob mice following leptin administration (Turner et al. 2013). Thus, the reduced cortical thickness in femurs of ob/ob mice could result from estrogen deficiency, leptin deficiency or combined deficiencies of the two hormones. The absence of an effect of ICI in leptin-treated mice indicates that the resulting increase in cortical thickness does not require estrogen signaling. However, increasing leptin levels while reducing the magnitude did not fully prevent bone loss in ovx rats (Burguera et al. 2001). Taken together, these observations suggest that leptin and estrogen have distinct as well as complementary effects on the skeleton and that both hormones may be required for optimal cortical thickness.
Estrogens play an important role in energy balance. Rodents typically exhibit excess weight gain following ovx due in part to hyperphagia (Clark & Tarttelin 1982). Pair-feeding ovx rodents to ovary-intact controls attenuates but does not completely prevent excess weight gain, suggesting that ovarian hormones also increase energy expenditure (Jiang et al. 2008). The effects of estrogen on energy balance are believed to occur through estrogen receptor-mediated signaling within the hypothalamus (Frank et al. 2014). ICI delivered into peripheral circulation may cross the blood–brain barrier and localize in the hypothalamus (Howell et al. 2000, Alfinito et al. 2008). As such, sc administered ICI could antagonize estrogen receptor-mediated pathways involved in energy metabolism in the central nervous system. In the present study, ICI did not alter the effects of leptin on food consumption or slow weight loss in ob/ob mice. Additionally, hypothalamic leptin gene therapy was shown to be effective in blocking the increase in appetite in rats following ovx (Torto et al. 2006, Ng et al. 2010). These findings suggest that a leptin-induced increase in estrogen levels is not required for leptin to suppress appetite. However, the dose rate of ICI used in the present study, although sufficient to block estrogen receptor-mediated actions on uterus and bone (Sibonga et al. 1998), was lower than the concentrations shown to induce hyperphagia and decrease energy expenditure in normal rats (Alfinito et al. 2008). It is therefore possible that higher doses of ICI are required to block the inhibitory actions of leptin-induced estrogen on appetite.
ob/ob mice develop morbid obesity. In contrast to adult WT mice, which were nearly weight stable throughout the one-month duration of our study, ob/ob mice gained weight. As anticipated, leptin treatment resulted in weight loss, which was due in part to lower abdominal WAT weight. We did not measure individual fat depots or lean mass but leptin treatment has been shown to result in a preferential reduction in total fat mass with minimal reduction in water and lean body mass (Rafael & Herling 2000). Total body weight and abdominal WAT weight, although lower than in untreated ob/ob mice, were still much higher at study termination in leptin-treated ob/ob mice than in WT mice. Similar to appetite, the absence of an effect of co-treatment with leptin and ICI on weight compared to leptin alone suggests that estrogen receptor signaling is largely dispensable for the leptin-mediated reduction in WAT and body weight. This conclusion is concordant with studies demonstrating the efficacy of leptin in preventing weight gain in ovx mice and rats (Torto et al. 2006, da Silva et al. 2014).
Adipose tissue produces a wide array of peptide hormones and cytokines, collectively referred to as adipokines (Ronti et al. 2006). Additionally, adipose tissue expresses aromatase activity and production of estrogens by WAT may contribute to the higher estrogen levels associated with obesity (Cleland et al. 1983, 1985, Magoffin et al. 1999, Liu et al. 2013). Uterine weight, a sensitive index of estrogen level, was positively associated with WAT weight in ob/ob mice (Turner et al. 2014). Leptin treatment increased uterine weight in ob/ob mice (Turner et al. 2014) and this response was blocked by ICI, indicating that ICI treatment was effective in blocking estrogen signaling, regardless of the tissue origin of the hormone.
In spite of drastic differences in abdominal WAT between calorically restricted mice (decreased) and mice with loss of function mutations in the gene for leptin or the gene for leptin receptor (increased), both conditions result in increased MAT (Bartell et al. 2011, Turner & Iwaniec 2011), a finding implicating leptin as an important negative regulator of MAT. This conclusion is supported by studies demonstrating that increasing leptin levels by sc administration of the hormone, direct delivery of the hormone into the hypothalamus, or by hypothalamic leptin gene therapy reduces MAT in ob/ob mice (Bartell et al. 2011, Turner et al. 2015). However, ovx rats and normal mice fed high fat diet have elevated MAT in spite of elevated leptin levels, and increasing hypothalamic leptin levels does not decrease MAT in normal and ovx rats (Martin & Zissimos 1991, Iwaniec et al. 2011, Jackson et al. 2011). This paradox is resolved if, similar to appetite and weight gain, the inhibitory effects of high levels of leptin on MAT are impaired by the development of leptin resistance (Sainz et al. 2015). In the present study, the reduction in MAT by sc administration of leptin to leptin-deficient mice was not prevented by co-administration with ICI. Taken together, these findings suggest that estrogen receptor signaling is not required for leptin to reduce MAT levels in ob/ob mice while leptin resistance may facilitate the increase in MAT associated with ovx.
Accurately modeling the diurnal fluctuations observed in serum leptin levels is challenging (Arble et al. 2011). In normal mice, leptin levels display a diurnal rhythm with a nadir in mid-morning and a nocturnal peak (Ahren 2000). In the present study, we administered leptin to leptin-deficient ob/ob mice by daily sc injection. This route of administration would be expected to result in much greater extremes in maximum and minimum levels of the hormone than occur physiologically. Nevertheless, once daily sc leptin treatment quickly decreased food intake to values similar to WT mice and maintained lower food intake through the remainder of study.
Bone balance is highly sensitive to changes in energy availability (Iwaniec & Turner 2016). Caloric restriction in adult rats, resulting in only 5% weight loss, had dramatic negative effects on bone metabolism, including reduced bone formation, increased osteoclast perimeter and reduced cancellous bone volume fraction (Turner & Iwaniec 2011). In contrast, increases in leptin levels in the hypothalamus of normal rats reduced food intake and body weight without inducing bone loss (Turner et al. 2015). However, a combination of caloric restriction (30%) and sc leptin treatment, while reducing MAT accumulation, did not prevent the detrimental skeletal changes associated with suppression of normal weight gain in rapidly growing mice (Devlin et al. 2016). These findings suggest that methods that improve leptin signaling have the potential to preserve bone mass during rapid weight loss in adults but are unlikely to compensate for inadequate energy availability during growth.
Adoptive transfer of leptin receptor-deficient db/db bone marrow cells into WT mice recapitulated the low bone formation observed in db/db mice without increasing food intake (Turner et al. 2013). Additionally, sc infusion of leptin revealed that the stimulatory effects of leptin on bone formation occur at leptin levels that have minimal effects on energy metabolism (Philbrick et al. 2017). These findings provide strong evidence that leptin acts peripherally to stimulate bone formation. However, the putative target cells in bone and precise mechanisms mediating the bone anabolic effects of leptin in ob/ob mice have not been identified.
It is likely that some of the profound abnormalities in bone metabolism in leptin-deficient ob/ob mice occur as a result of comorbidities and hormonal changes. Comorbidities include hypogonadism, hyperphagia, impaired thermoregulation and hyperglycemia. Alterations in bone regulating hormones and growth factors include increased corticosteroid levels, and low levels of sex steroids, growth hormone and IGF1 (Saito & Bray 1983, Ozata et al. 1999, Segev et al. 2007, Turner et al. 2014). Additionally, hyperparathyroidism is associated with leptin deficiency in humans (Ozata et al. 1999). In the present study, we demonstrate that ovarian hormones play a limited role in mediating the skeletal actions of leptin. In prior studies, we showed that hyperphagia, hyperglycemia and impaired thermoregulation, typically observed in ob/ob mice, actually attenuate skeletal abnormalities in these animals (Turner et al. 2014). Taken together, these findings indicate that leptin plays an important role in regulating bone growth, maturation and turnover but further research is required to understand the full range of actions by the hormone.
In summary, the profound abnormalities in metabolism in ob/ob mice, including hyperphagia and morbid obesity were attenuated or reversed following combination treatment with leptin and the estrogen receptor antagonist ICI. Similarly, leptin + ICI re-established compensatory increases in bone growth and near normal bone turnover, without restoring normal uterine weight. The increase in osteoblast-lined bone perimeter and bone formation in leptin + ICI treated ob/ob mice was accompanied by a reduction in MAT, suggesting that treatment promoted the differentiation of stromal cells to osteoblasts at the expense of adipocytes. Finally, the limited differences in skeletal endpoints between ob/ob mice treated with leptin compared to ob/ob mice treated with leptin + ICI indicate that estrogen receptor signaling is dispensable for the positive actions of leptin on bone and that estrogen may attenuate leptin-induced bone growth.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from the NIH (AR060913), NASA (NNX12AL24G) and USDA (38420-17804).
Author contribution statement
Study design was done by R T T, K A P, A J B and U T I. Study execution was done by K A P. Data collection was done by K A P, A K and R T T. Data analysis was done by A J B. Data interpretation was done by R T T, K A P, A J B and U T I. Drafting of manuscript was done by R T T and U T I. Revising manuscript content was done by K A P, A J B, R T T and U T I. Approval of the final version of manuscript was done by R T T, K A P, A K, A J B and U T I. U T I takes responsibility for the integrity of the data.
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