Daily leptin blunts marrow fat but does not impact bone mass in calorie-restricted mice

in Journal of Endocrinology

Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation, suggesting that hypoleptinemia may be a link between negative energy balance, bone marrow fat accumulation, and impaired skeletal acquisition. In that case, treating mice with leptin during caloric restriction (CR) should reduce marrow adipose tissue (MAT) and improve bone mass. To test this hypothesis, female C57Bl/6J mice were fed a 30% CR or normal (N) diet from 5 to 10 weeks of age, with daily injections of vehicle (VEH), 1mg/kg leptin (LEP1), or 2mg/kg leptin (LEP2) (N=6–8/group). Outcomes included body mass, body fat percentage, and whole-body bone mineral density (BMD) via peripheral dual-energy X-ray absorptiometry, cortical and trabecular microarchitecture via microcomputed tomography (μCT), and MAT volume via μCT of osmium tetroxide-stained bones. Overall, CR mice had lower body mass, body fat percentage, BMD, and cortical bone area fraction, but more connected trabeculae, vs N mice (P<0.05 for all). Most significantly, although MAT was elevated in CR vs N overall, leptin treatment blunted MAT formation in CR mice by 50% vs VEH (P<0.05 for both leptin doses). CR LEP2 mice weighed less vs CR VEH mice at 9–10 weeks of age (P<0.05), but leptin treatment did not affect body fat percentage, BMD, or bone microarchitecture within either diet. These data demonstrate that once daily leptin bolus during CR inhibits bone marrow adipose expansion without affecting bone mass acquisition, suggesting that leptin has distinct effects on starvation-induced bone marrow fat formation and skeletal acquisition.

Abstract

Starvation induces low bone mass and high bone marrow adiposity in humans, but the underlying mechanisms are poorly understood. The adipokine leptin falls in starvation, suggesting that hypoleptinemia may be a link between negative energy balance, bone marrow fat accumulation, and impaired skeletal acquisition. In that case, treating mice with leptin during caloric restriction (CR) should reduce marrow adipose tissue (MAT) and improve bone mass. To test this hypothesis, female C57Bl/6J mice were fed a 30% CR or normal (N) diet from 5 to 10 weeks of age, with daily injections of vehicle (VEH), 1mg/kg leptin (LEP1), or 2mg/kg leptin (LEP2) (N=6–8/group). Outcomes included body mass, body fat percentage, and whole-body bone mineral density (BMD) via peripheral dual-energy X-ray absorptiometry, cortical and trabecular microarchitecture via microcomputed tomography (μCT), and MAT volume via μCT of osmium tetroxide-stained bones. Overall, CR mice had lower body mass, body fat percentage, BMD, and cortical bone area fraction, but more connected trabeculae, vs N mice (P<0.05 for all). Most significantly, although MAT was elevated in CR vs N overall, leptin treatment blunted MAT formation in CR mice by 50% vs VEH (P<0.05 for both leptin doses). CR LEP2 mice weighed less vs CR VEH mice at 9–10 weeks of age (P<0.05), but leptin treatment did not affect body fat percentage, BMD, or bone microarchitecture within either diet. These data demonstrate that once daily leptin bolus during CR inhibits bone marrow adipose expansion without affecting bone mass acquisition, suggesting that leptin has distinct effects on starvation-induced bone marrow fat formation and skeletal acquisition.

Introduction

Bone marrow adipose tissue (MAT) is a complex and dynamic depot that likely includes both constitutive and regulated cell populations (Devlin & Rosen 2015, Scheller et al. 2015). MAT accumulation is a normal component of skeletal aging in healthy individuals, but is also observed in metabolic disease, including anorexia nervosa, diabetes, and osteoporosis, and in skeletal unloading, including paralysis and bedrest (Yeung et al. 2005, Bredella et al. 2009, Trudel et al. 2009, Ecklund et al. 2010, Tuljapurkar et al. 2011, Baum et al. 2012, Schwartz et al. 2013). As has been widely noted, MAT may influence bone mass via interactions with adjacent osteoblasts in the marrow, as adipocytes and osteoblasts derive from a common mesenchymal progenitor (Muruganandan et al. 2009, Bianco et al. 2010). Although bone–fat interactions involve multiple hormones and growth factors, several lines of evidence suggest a role for the adipokine leptin in mediating the balance between bone and fat in the endosteal niche. First, leptin promotes the differentiation of bone marrow mesenchymal stem cells in vitro to the osteoblast rather than the adipocyte lineage, leading to the hypothesis that low leptin could lead to a relative increase in fat vs bone cells (Thomas et al. 1999). Secondly, leptin replacement in hypoleptinemia decreases skeletal fragility in animal models (Cornish et al. 2002) and increases bone formation markers in humans (Welt et al. 2004). Finally, the adipokine leptin falls in starvation, raising the possibility that hypoleptinemia contributes to marrow fat expansion and impaired skeletal acquisition (Ahima et al. 1996, Boden et al. 1996). Interactions between bone and fat may be particularly important in young, rapidly growing animals, as CR in young mice is associated with both low bone mass and high marrow adiposity (Devlin et al. 2010), whereas CR in older mice has more modest effects on bone and may not alter marrow adiposity (Hamrick et al. 2008, Tatsumi et al. 2008). Thus, here we test whether daily leptin bolus during caloric restriction (CR) reduces marrow fat and improves bone mass, using the established system of CR in young, rapidly growing mice to model human energy restriction (Devlin et al. 2010).

Materials and methods

Mouse model and experimental protocol

This study used female C57Bl/6J mice (Jackson Laboratories), studied from 5 to 10 weeks of age. Mice were weighed at baseline and assigned to groups of equivalent body mass (mean±s.d.). Mice were housed individually and fed a normal (N) diet ad libitum (Research Diets 12450B, 10% kcal/fat) or a 30% CR diet with supplemental micronutrients, which provided equivalent vitamins and minerals when fed at 70% of caloric intake ad libitum (Research Diets D10012703, 10% kcal/fat). Food allotments were placed into CR cages daily at 18:00h. Body mass was measured daily on a digital scale. Body length was measured at 5 and 10 weeks of age. Mice were killed by CO2 inhalation at approximately 10:00h. Experimental protocols were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.

Leptin administration

At 18:00h daily, all mice received subcutaneous injection of recombinant murine leptin (R&D Systems) at 1mg/kg/day (LEP1), leptin at 2mg/kg/day (LEP2), or vehicle (VEH) (N=6–8/group). Dosages were adjusted daily based on the body mass. We used a single late-day bolus in order to replicate the murine leptin peak that occurs in the dark cycle, during the period of greatest food intake (Ahima et al. 1996, Ahren 2000). The two doses tested, 1 or 2mg/kg/day, correspond to roughly 20–40μg leptin/day for a 20g mouse and were intended to produce serum leptin levels in the physiological range of 4–8ng/mL in C57Bl/6J mice, as previously reported (Ahima et al. 1996, Ahren 2000, Surwit et al. 2000).

Peripheral dual-energy X-ray absorptiometry

In vivo measurements of whole-body (excluding the head) bone mineral density (BMD, g/cm2), bone mineral content (BMC, g), and body composition (% body fat) were obtained at 5 and 10 weeks of age using peripheral dual-energy X-ray absorptiometry (pDXA, PIXImus II, GE Lunar Corporation, Madison, WI, USA), as previously described (Bouxsein et al. 2009).

Serum hormones

At the time of killing, blood was collected by cardiac puncture to measure serum leptin by ELISA (Crystal Chem, Downers Grove, IL, USA) (Devlin et al. 2010). The assay sensitivity was 0.2–12.8ng/mL.

Specimen collection and preparation

After the animals were killed, gonadal white adipose depots were dissected and weighed. Femurs, tibiae, and fifth lumbar vertebrae were excised and cleaned. The right femur and L5 vertebral body were wrapped in saline-soaked gauze and frozen at −20°C for imaging analysis (Devlin et al. 2010). The left tibia was prepared for histology in 70% ethanol at 4°C until processing. The right tibia was fixed in 10% neutral buffered formalin at 4°C overnight and stored in PBS for osmium tetroxide staining (Scheller et al. 2014).

Trabecular and cortical bone morphology by microcomputed tomography

Bone microarchitecture of the femoral midshaft, distal femur, and vertebral body was measured via high-resolution microcomputed tomography (μCT40, Scanco Medical, Brüttisellen, Switzerland), as previously des­cribed (Glatt et al. 2007). Scans were acquired using a 12μm3 isotropic voxel size, 70kVP, 114mA, and 200ms integration time, and were subjected to Gaussian filtration and segmentation. In the distal femur, transverse CT slices were evaluated beginning 360μm proximal to the growth plate, extending 1800μm proximally. At the femoral mid-diaphysis, transverse μCT slices spanning 600μm were obtained, whereas in the fifth lumbar vertebra, transverse CT slices spanning the entire vertebra, excluding growth plates, were obtained (2400–3000μm). Thresholds of 287, 708, and 365mg hydroxyapatite (HA)/cm3 were used to segment the bone from the soft tissue for the distal femur, femoral diaphysis, and vertebral body, respectively. Terminology and units followed the recommendations of the American Society for Bone and Mineral Research (Bouxsein et al. 2010).

Histology and quantitative histomorphometry

To examine bone formation rates, calcein (15mg/kg) was injected intraperitoneally at 9 and 2 days before killing the animals. Undecalcified bones were dehydrated and cleared on a tissue processor using an alternating vacuum/pressure cycle and, once cleared, infiltrated under vacuum for 3 days at 4°C with 85% methylmethacrylate (MMA) and 15% dibutyl phthalate (DP). Following infiltration, bones were embedded in 85% MMA, 15% DP, and 2.5% benzoyl peroxide (catalyst), and polymerized in a radiant heat oven at 37°C. Once polymerized, bones were cut on a Leica 2265 microtome using a 160 mm D-profile tungsten-carbide knife (Dorn and Hart, Loxley, AL, USA). Unstained sections for dynamic histomorphometry were coverslipped directly with a UV inert mountant. Sections for static histomorphometry were deplastified and stained with toluidine blue (Fisher), pH 4.2. Microscopic analysis of static and dynamic parameters was performed using an Olympus BX40 microscope interfaced with the Osteomeasure system software and hardware (Osteometrics, Atlanta, GA, USA). All static parameters were measured on trabecular bone beginning immediately under the growth plate excluding endosteal surfaces. The field size for static parameters was 290×290μm, and the area measured under the growth plate extended 580μm under the growth plate where most of the trabecular bone is located. Dynamic parameters of bone formation were read similarly using a Nikon Labophot scope equipped with epifluorescence to visualize pulse-labeled calcein on the trabecular surfaces. Mineralizing surface was calculated using double labels plus half the single-labeled surface. The field size for dynamic parameters was 350×350μm and extended 700μm under the growth plate. Terminology and units followed the recommendations of the American Society for Bone and Mineral Research (Dempster et al. 2013).

Osmium tetroxide staining

Left tibiae were fixed in 10% neutral buffered formalin at 4°C and decalcified in EDTA. Marrow fat was stained with a 1:1 solution of 2% aqueous osmium tetroxide (OsO4) and 5% potassium dichromate (Turello et al. 1984, Fretz et al. 2010, Scheller et al. 2014). Fat volume in the proximal and distal tibia medullary compartments was quantified using high-resolution microcomputed tomography as described previously (μCT40, Scanco Medical, Brüttisellen, Switzerland). For the proximal tibia, CT slices were evaluated beginning 1mm below the growth plate and extending 2400μm distally. In the distal tibia, a 2400μm region was evaluated beginning at the tibiofibular junction and extending distally. The medullary canal was identified by manually tracing the endocortical border. Osmium-stained marrow fat was segmented from the adjacent marrow using a fixed threshold (1134mg HA/cm3). Adipose volume (mm3), marrow volume (mm3), and adipose/marrow volume fraction (%) were measured.

Statistical analyses

Standard descriptive statistics were computed for all outcome variables, and data checked for normality. We used a General Linear Model in SPSS 22 (IBM) to test for the effect of diet, leptin, and leptin–diet interactions on body composition and bone variables. All tests were two tailed, with the significance level for major effects set at α=0.05. If there was an overall significant effect of diet, leptin, or leptin–diet interaction for a given variable, we performed post hoc Tukey tests to identify pairwise differences. As long bone cortical cross-sectional geometry is influenced by body mass, we also adjusted femoral cortical variables (Ct.Th, Ct.Ba, Ct.TA, Imax, Imin, pMOI) by regressing these variables against body mass and testing for significance using the residuals (Lang et al. 2005).

Results

CR mice have lower body mass, body fat, serum leptin, and BMD

Body mass was lower in CR vs N mice from 6 to 10 weeks of age (P<0.0001), and in CR LEP2 vs CR VEH mice at 9–10 weeks of age (P<0.03 for both, Fig. 1A). From 5 to 10 weeks of age, the total body fat mass increased by 216% in N mice vs 6% in CR mice (P<0.03); within the CR group, fat mass in VEH mice increased by 24% despite overall weight loss, whereas LEP1 and LEP2 maintained and lost body fat, respectively (P<0.04, Fig. 1B). Lean mass decreased in CR by 22% and increased in N mice by 9.5% (P<0.001), Fig. 1D). At 10 weeks of age, CR mice had shorter femurs, smaller bodies, and smaller perigonadal fat pads compared with N (P<0.0001 for all, Table 1). Fasting serum leptin, measured 16h after the final daily injection for all mice and the final daily feeding for CR, was markedly lower in CR vs N mice (2.5 vs 10.5ng/mL, P<0.05) and did not differ across leptin treatments within either diet (Fig. 1D). BMD and BMC at 10 weeks of age and the gain in BMC from 5 to 10 weeks of age were lower in CR vs N overall (P<0.0004, Fig. 1E and Table 1), but did not differ within either diet across leptin treatments.

Figure 1
Figure 1

(A) CR weighed less than N at 6–10 weeks of age, and CR LEP2 weighed less vs CR VEH at 9–10 weeks of age (P<0.0001 for diet at 6–10 weeks, P<0.03 for dose at 9 and 10 weeks). (B) From 5 to 10 weeks of age, CR gained less body fat (g) vs N (P<0.0001 for diet and P=0.023 for dose), and within CR, LEP1 and LEP2 gained less body fat vs VEH (P<0.04 for both). (C) From 5 to 10 weeks of age, CR lost lean mass, whereas N gained lean mass (P<0.0001 for diet) (D) At 10 weeks of age, CR had markedly lower leptin vs N irrespective of treatment group (P<0.0001 for diet). Daily leptin injections at 18:00h did not alter fasting serum leptin ~16h postinjection, consistent with short half-life. (E) From 5 to 10 weeks of age, CR exhibited smaller gains in BMC vs N mice irrespective of leptin treatment (P=0.001 for diet).

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0473

Table 1

Body size and body composition at 10 weeks of age in female C57Bl/6J mice fed 30% CR diet or N diet ad libitum from 5 to 10 weeks of age with daily injection of leptin (1–2 mg/kg/day) or vehicle.

CRN
Vehicle1mg/kg/day2mg/kg/dayVehicle1mg/kg/day2mg/kg/dayOverall P-value
Means.d.Means.d.Means.d.Means.d.Means.d.Means.d.DietLeptinDiet–Leptin
ANL (mm)80.75a2.4981.50a1.4179.17a1.3385.571.9986.881.5586.632.33<0.0001NSNS
Femur length (mm)12.50a0.2012.62a0.0812.51a0.2813.440.1913.530.3213.600.24<0.0001NSNS
Perigonadal WAT (g/g body weight)0.073a0.0460.036a0.0180.037a0.0410.3240.1340.2950.1520.1960.089<0.0001NSNS
Lean mass (g)9.91a0.649.64a0.238.98a0.5913.180.9513.330.7613.250.78<0.0001NSNS
Fat mass (g)2.60a0.352.10a,b0.131.91a,b0.165.101.474.311.363.550.86<0.00010.008NS
WB BMD (g/cm2)0.042a0.0020.042a0.0020.043a0.0010.0460.0020.0450.0010.0440.001<0.0001NSNS
WB BMC (g)0.283a0.0220.300a0.0250.313a0.0180.3200.0240.3240.0220.3300.0220.0004NSNS

Significant overall difference in CR vs N mice with leptin treatments pooled

Significant difference from vehicle treated within diet.

ANL, anus-to-nose length; WAT, white adipose tissue (g/g body weight); WB BMD, whole-body bone mineral density; WB BMC, whole-body bone mineral content.

Leptin treatment did not affect trabecular or cortical microarchitecture

In the distal femur trabecular bone, CR mice had lower structural model index (SMI) and higher Conn.D vs N (P<0.0001 for both), but Tb.BV/TV, Tb.N, and Tb.Th did not differ between CR and N mice (Fig. 2A and Table 2). There were no differences in trabecular bone variables across leptin treatments within either diet group (Fig. 2A and Table 2).

Figure 2
Figure 2

(A) Distal femur Tb.BV/TV (%) did not differ by diet or by leptin treatment. (B) Ct.TA (mm2), (C) Ct.BA/TA (%), and (D) Ct.pMOI were lower in CR vs N before body mass adjustment (P<0.0001 for diet), but did not differ by leptin treatment.

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0473

Table 2

Cortical and trabecular microarchitecture at 10 weeks of age in female C57Bl/6J mice fed 30% CR diet or N diet ad libitum from 5 to 10 weeks of age with daily injection of leptin (1–2 mg/kg/day) or vehicle.

CRN
Vehicle1mg/kg/day2mg/kg/dayVehicle1mg/kg/day2mg/kg/dayOverall P-value
Means.d.Means.d.Means.d.Means.d.Means.d.Means.d.DietLeptinDiet–Leptin
Distal femur trabecular bone
Tb.N (/mm)4.270.624.380.854.140.534.230.244.160.263.980.23NSNSNS
Tb.Th (mm)0.0470.0010.0470.0020.0450.0010.0490.0020.0470.0020.0470.003NSNSNS
Tb.Sp (mm)0.2360.0410.2330.0640.2400.0360.2310.0160.2370.0180.2470.017NSNSNS
Conn.D (/mm)164.2a42.5166.9a55.0140.9a45.7115.516.7102.317.696.611.7<0.0001NSNS
SMI2.40a0.262.34a0.392.54a0.252.730.112.840.182.870.13<0.0001NSNS
Midshaft femur cortical bone
BA0.4800.0310.4790.0250.4650.0330.5710.0300.5770.0320.5740.038<0.0001NSNS
Ct.Th (mm)0.111a,b0.0060.113a,b0.0050.109a,b0.0050.1310.0070.1310.0090.1310.008<0.0001NSNS
Imax (mm4)0.142a,b0.0200.141a,b0.0160.135a,b0.0210.1750.0100.1830.0130.1790.019<0.0001NSNS
Imin (mm4)0.077a,b0.0090.071a,b0.0080.071a,b0.0090.0910.0080.0940.0080.0910.009<0.0001NSNS
Lumbar vertebra trabecular bone
BV/TV (%)24.531.5024.771.5325.211.6826.151.8524.031.7623.00c2.85NSNS0.036
Tb.N (/mm)5.34a0.275.27a0.115.29a0.165.000.134.880.274.760.30<0.0001NSNS
Tb.Th (mm)0.048a0.0010.049a0.0020.048a0.0020.0540.0020.0520.0020.0510.002<0.0001NSNS
Tb.Sp (mm)0.180a0.0110.180a0.0040.180a0.0060.1920.0080.1980.0130.2040.013<0.0001NSNS
Conn.D (/mm)226.1a26.5197.1a16.5210.5a19.0200.312.6195.429.7191.120.00.022NSNS
SMI0.880.150.820.200.750.220.730.170.930.190.970.27NSNS0.044

Significant overall difference in CR vs N mice with leptin treatments pooled

No longer significant after adjustment for body mass

Significant difference from vehicle treated within diet.

At the femoral midshaft, cortical bone cross-sectional geometric properties, including Ct.Th, Ct.BA, Ct.TA, Imax, Imin, and pMOI, were lower in CR vs N (P<0.0001 for all, Fig. 2B, C, D and Table 2), but these differences no longer reached statistical significance after body mass adjustment (Lang et al. 2005). There were no differences in cross-sectional geometric properties across leptin treatments within either diet group.

In the fifth lumbar vertebra, N LEP2 mice had lower Tb.BV/TV vs N VEH (P=0.033, Table 2). CR mice had higher Tb.N and Conn.D, and lower Tb.Th and Tb.Sp vs N (P<0.03 for all, Table 2), but there were no differences across leptin treatments within each diet.

Leptin blunted MAT expansion in CR mice

Daily leptin bolus during CR partially suppressed the expansion of MAT observed in CR VEH mice (Fig. 3A). In the proximal tibia, adipose volume/marrow volume as quantified by μCT scanning of bones stained with osmium tetroxide was higher in CR vs N overall (P<0.0001), but 50% lower in CR LEP1 and CR LEP2 vs CR VEH (P<0.05 for both, Fig. 3B). Quantitative histomorphometry confirmed this pattern, with N.Ad/T.Ar higher in CR compared with N overall (P<0.002), but lower in CR LEP1 and CR LEP2 compared with CR VEH (P=0.046, Table 3). BV/TV (%) of the proximal tibia did not differ in CR vs N (Table 3). N.Ob/T.Ar and N.Ob/BS were lower in CR compared with N overall (P<0.02 for both, Table 3), but did not differ across leptin treatments within either diet. Dynamic markers of bone formation, including sLS/BS, dLS/BS, MAR, MS/BS, and BFR/BS, were 36–108% lower in CR vs N overall, but did not differ across leptin treatments within either diet (P<0.0001 for all, Table 3).

Figure 3
Figure 3

(A) Leptin treatment during CR blunted MAT expansion compared with CR VEH, as indicated by extent of OsO4 staining (shown in dark gray). (B) Proximal tibia adipose volume/marrow volume in OsO4 stained bones was higher in CR vs N, and 50% lower in CR LEP1 and CR LEP2 vs CR VEH (P<0.0001 for diet, P=0.016 for dose).

Citation: Journal of Endocrinology 229, 3; 10.1530/JOE-15-0473

Table 3

Number of osteoblasts, osteoclasts, and adipocytes at 10 weeks of age in the proximal tibia of female C57Bl/6J mice fed 30% CR diet or N diet ad libitum from 5 to 10 weeks of age with daily injection of leptin (1–2 mg/kg/day) or vehicle.

CRN
Vehicle1mg/kg/day2mg/kg/dayVehicle1mg/kg/day2mg/kg/dayOverall P-value
Means.d.Means.d.Means.d.Means.d.Means.d.Means.d.DietLeptinDiet–Leptin
BV/TV (%)21.32.920.83.719.12.420.94.921.32.920.83.7NSNSNS
N.Ob/T.Ar (/mm2)79.9a53.061.4a38.152.2a8.9100.242.298.686.8118.146.70.013NSNS
N.Ob/BS (/mm)6.7a4.55.3a3.54.7a1.210.04.410.37.712.84.70.001NSNS
N.Oc/T.Ar (/mm2)20.211.118.18.121.25.221.98.317.98.918.97.2NSNSNS
N. Oc/BS (/mm)1.70.81.50.71.90.62.21.02.00.72.00.6NSNSNS
N.Ad/T.Ar (/mm2)112.0a52.285.4a,b46.860.5a,b20.626.520.821.316.415.34.2<0.0020.046NS
sLS/BS (%)13.7a5.2414.28a3.8913.62a2.8418.344.0622.204.9420.936.85<0.0001NSNS
dLS/BS (%)9.63a4.7510.86a4.7112.76a3.2718.453.6516.374.9016.066.79<0.0001NSNS
MS/BS (%)16.5a6.118.0a5.419.6a3.727.63.427.54.826.55.0<0.0001NSNS
MAR (µm/day)1.39a0.281.31a0.241.36a0.231.710.161.890.231.900.39<0.0001NSNS
BFR/BS (mm3/mm2*year)86.5a41.085.3a29.998.6a30.2172.227.2199.336.7183.851.5<0.0001NSNS

Significant overall difference in CR vs N mice with leptin treatments pooled

Significant difference from vehicle treated within diet.

Discussion

In this study, we tested the effects of a daily leptin bolus of 1–2mg/kg/day during CR in young, rapidly growing mice. We hypothesized that leptin treatment would attenuate the deleterious effects of CR on bone mass and bone marrow adiposity. To test this hypothesis, we evaluated BMD, cortical and trabecular bone properties, and marrow adipose expansion in female C57Bl/6J mice from 5 to 10 weeks of age. VEH-treated CR mice lost body mass, particularly lean mass, and exhibited smaller gains in body fat and BMD during the experiment compared with VEH-treated N controls. The skeletal phenotype of VEH-treated CR mice included lower whole-body BMD and lower cross-sectional geometric properties in the midshaft femur before adjustment for body mass, with no effects observed on the trabecular bone microarchitecture. Static and dynamic histomorphometry in the proximal tibia showed that the bones of CR mice had fewer osteoblasts, more adipocytes, and lower indices of bone formation compared with N mice.

Our first hypothesis, that leptin treatment during CR would increase bone mass acquisition, was not supported by the data. Daily leptin injection of 1–2mg/kg/day did not increase BMD, alter cortical or trabecular bone architecture, or increase bone formation indices in CR mice, nor did it alter skeletal phenotype in N mice fed ad libitum. There was support for our second hypothesis that leptin would blunt the increase in marrow adiposity observed with exposure to a CR diet. Specifically, VEH-treated CR mice had a fourfold increase in marrow adiposity compared to VEH-treated N mice. Leptin treatment reduced the CR-induced expansion of marrow adipose in the proximal tibia by 50%, although the MAT volume was still markedly higher than in N mice. These data support the hypothesis that hypoleptinemia contributes to MAT expansion during CR.

Comparison to prior studies of leptin and bone

Hypoleptinemia is associated with low bone mass in humans with anorexia nervosa (Hebebrand et al. 1997) and in animal models (Devlin et al. 2010). The skeletal effects of leptin are complex and include both central nervous system pathways via the hypothalamus and peripheral pathways involving direct effects of leptin on bone cells. Studies in wild-type mice reported lower trabecular bone volume fraction in the vertebra following intracerebroventricular (ICV) leptin infusion (Ducy et al. 2000, Takeda et al. 2002, Sato et al. 2007). By contrast, in the hypoleptinemic ob/ob mouse, ICV leptin improved bone formation and reduced MAT (Hamrick et al. 2007, Bartell et al. 2011), and hypothalamic leptin gene therapy increased femur length, total femur volume, and circulating osteocalcin but reduced trabecular bone volume in the distal femur and vertebra (Iwaniec et al. 2007, 2009, Kalra et al. 2009).

The peripheral or direct effects of leptin on bone are similarly heterogeneous. The finding that leptin receptor (Lepr) deletion in osteoblasts had no effect on murine skeletal phenotype implied little direct effect of leptin on osteoblasts (Shi et al. 2008). However, Zhou and coworkers recently reported that bone marrow mesenchymal stromal cells expressing the leptin receptor can differentiate into the bone, cartilage, and adipocytes, both in vivo and in vitro, and showed by fate mapping that in adult bone marrow, these leptin receptor-positive cells represent the primary source of bone and adipocytes (Zhou et al. 2014). In vitro, leptin increased osteoblast and chondrocyte proliferation, and inhibited osteoclast formation, and in adult male wild-type mice, peripheral injection of leptin (43µg/day) increased the bone strength, suggesting anabolic effects of leptin on cortical bone (Cornish et al. 2002). Leptin treatment in mice subjected to 40% CR prevented the CR-induced decrease in longitudinal bone growth (Gat-Yablonski et al. 2004). In the ob/ob mouse, peripheral leptin improved bone mass and bone formation rate and reduced MAT (Steppan et al. 2000, Hamrick et al. 2005, Turner et al. 2013). Continuous infusion of supraphysiological leptin doses (100 or 200mg/day) in female rats increased serum leptin by 14- to 33-fold and decreased body mass, but while the 100mg/day dose led to lower whole-body BMD, femoral cortical bone volume, femoral bone strength, and femoral polar moment of inertia, the higher dose did not, suggesting that bone loss was prevented despite weight loss (Stunes et al. 2012). High-dose leptin (2.5mg/kg 2×/day) also prevented the fasting-induced decrease in osteocalcin in BALB/c males (Goldstone et al. 2002).

Taken together, these studies suggest that both centrally and peripherally administered leptin can be anabolic, neutral, or deleterious to bone mass. Although some of this variability likely reflects the different background strains, dosages, routes of administration, sexes, and ages at onset and offset of treatment used in different studies, it appears that in general, leptin is anabolic to bone in hypoleptinemic animals, but has less effect in leptin-replete animals unless delivered at supraphysiological doses (Kawai et al. 2009). In this study, the lack of skeletal effects of leptin likely reflects an insufficient dose and/or route of administration (see below).

Comparison to prior studies of leptin and MAT

Previous studies have demonstrated that leptin decreases MAT in genetic models of hypoleptinemia (Hamrick et al. 2005, Turner et al. 2013) and in leptin-replete wild-type rats (Hamrick et al. 2007). Humans with anorexia nervosa similarly exhibited attenuation of marrow adiposity with weight recovery (Fazeli et al. 2012). The inverse relationship of leptin and marrow adiposity may reflect direct as well as indirect mechanisms. Bone marrow adipocytes, osteoblasts, and myocytes derive from a common mesenchymal progenitor (Bianco et al. 2008, 2010, Zhou et al. 2014), and leptin directly promotes osteoblastogenesis and suppresses adipogenesis, such that hypoleptinemia could both increase marrow adiposity and decrease bone mass (Muruganandan et al. 2009, Evans et al. 2011). In addition, there may be cross talk between leptin, estrogen, and the GH–IGF1 axis, both of which are suppressed in CR in humans and animal models (Mobbs et al. 2001, Devlin et al. 2010, Misra & Klibanski 2011). Postmenopausal estrogen deficiency in women is associated with MAT expansion, and estrogen replacement increases serum leptin and decreases marrow adiposity (Syed et al. 2008, Limonard et al. 2015). GH deficiency leads to expansion of MAT that is attenuated by GH replacement in dwarf (dw/dw) rats (Gevers et al. 2002) and in hypophysectomized mice and rats (Menagh et al. 2010). In humans, vertebral MAT is inversely associated with circulating IGF1 (Bredella et al. 2010). Thus, in addition to direct action on osteoblast and adipocyte progenitors, leptin replacement during CR may decrease MAT by increasing GH and/or estrogen levels (Casanueva & Dieguez 1999, Ahima et al. 2000, Chou et al. 2011), a possibility that should be tested in future studies. Another possibility is that glucocorticoids are key to MAT expansion. A new study of male and female C57BL/6J mice exposed to CR from 9 to 15 weeks found CR-induced hypoleptinemia and loss of WAT in males but not females (Cawthorn et al. 2016). Further, CR in rabbits from 6–13 or 15–22 weeks of age induced hypoleptinemia, lower bone mass, and loss of WAT, but not MAT expansion (Cawthorn et al. 2016). These intriguing findings demonstrate that bone loss, hypoleptinemia, and MAT expansion are not inevitable consequences of CR and may occur independently of one another depending on age, sex, and species. The authors suggest that a CR-induced increase in glucocorticoids observed in mice but not rabbits may be the driver of MAT expansion (Cawthorn et al. 2016).

Limitations and future directions

Our study has several limitations that should be addressed in future work. First, and most importantly, we used a leptin dosage of 1–2mg/kg/day, or 20–40µg leptin/day, in a single daily bolus timed to coincide with the nocturnal peak in leptin following food intake (Ahima et al. 1996, Ahren 2000). This dosage and administration route may have induced transiently normal or high leptin levels but were insufficient to achieve sustained normalization of serum leptin in CR mice, as measured 16h following injection. Leptin has a short half-life, and it is clear that higher doses given via multiple daily injections or continuous leptin infusion will be needed to fully correct hypoleptinemia in CR animals. For example, daily leptin injection at 8µg/g was sufficient to reverse inhibition of longitudinal bone growth in CR, although serum leptin levels were not reported (Gat-Yablonski et al. 2004). Secondly, these data reflect only female mice, and there may be sex differences in the response to CR and/or effects of leptin on the bone and MAT. Thirdly, we studied mice during a single interval of 5–10 weeks of age. The effects of CR on the bone mass show marked age-related variation (Hamrick et al. 2008, Tatsumi et al. 2008, Devlin et al. 2010), and its effects on leptin levels and MAT expansion may also vary with age. Fourthly, here we focused solely on leptin and did not assess other bone-active hormones such as adiponectin or osteocalcin that are known to change in CR (Cawthorn et al. 2014) and to contribute to skeletal homeostasis in humans (Gravenstein et al. 2011). Finally, we studied only a single strain, C57Bl/6J, and it is clear that the relationships between the bone mass and MAT vary tremendously across strains (Fazeli et al. 2013, Scheller et al. 2015). Thus, future studies should include males; incorporate different ages and durations of CR exposure; consider additional hormonal factors; and include strains such as C3H/HeJ, which exhibits both high bone mass and high MAT on diet ad libitum (Motyl & Rosen 2011, Scheller et al. 2015).

Conclusion

In this study, we found that once daily leptin treatment of 1–2mg/kg/day is sufficient to blunt CR-induced MAT expansion, but does not alter the serum leptin level or cortical or trabecular bone architecture or bone formation in young female C57Bl/6J mice. These results suggest that bone MAT and bone tissue may have different sensitivities to small and/or transient changes in circulating leptin level. Such subtle changes may be sufficient to reduce MAT without inducing a corresponding increase in bone mass during CR in young female mice.

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 the National Institutes of Health (RC1AR058389, R24DK092759, S10RR017868, and F32HD060419).

Acknowledgments

The authors thank Mark Horowitz, Yale School of Medicine, New Haven, Connecticut, for assistance with bone histomorphometry and osmium tetroxide staining.

References

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    (A) CR weighed less than N at 6–10 weeks of age, and CR LEP2 weighed less vs CR VEH at 9–10 weeks of age (P<0.0001 for diet at 6–10 weeks, P<0.03 for dose at 9 and 10 weeks). (B) From 5 to 10 weeks of age, CR gained less body fat (g) vs N (P<0.0001 for diet and P=0.023 for dose), and within CR, LEP1 and LEP2 gained less body fat vs VEH (P<0.04 for both). (C) From 5 to 10 weeks of age, CR lost lean mass, whereas N gained lean mass (P<0.0001 for diet) (D) At 10 weeks of age, CR had markedly lower leptin vs N irrespective of treatment group (P<0.0001 for diet). Daily leptin injections at 18:00h did not alter fasting serum leptin ~16h postinjection, consistent with short half-life. (E) From 5 to 10 weeks of age, CR exhibited smaller gains in BMC vs N mice irrespective of leptin treatment (P=0.001 for diet).

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    (A) Distal femur Tb.BV/TV (%) did not differ by diet or by leptin treatment. (B) Ct.TA (mm2), (C) Ct.BA/TA (%), and (D) Ct.pMOI were lower in CR vs N before body mass adjustment (P<0.0001 for diet), but did not differ by leptin treatment.

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    (A) Leptin treatment during CR blunted MAT expansion compared with CR VEH, as indicated by extent of OsO4 staining (shown in dark gray). (B) Proximal tibia adipose volume/marrow volume in OsO4 stained bones was higher in CR vs N, and 50% lower in CR LEP1 and CR LEP2 vs CR VEH (P<0.0001 for diet, P=0.016 for dose).

  • AhimaRSPrabakaranDMantzorosCQuDLowellBMaratos-FlierEFlierJS1996Role of leptin in the neuroendocrine response to fasting. Nature382250252. (doi:10.1038/382250a0)

    • Search Google Scholar
    • Export Citation
  • AhimaRSSaperCBFlierJSElmquistJK2000Leptin regulation of neuroendocrine systems. Frontiers in Neuroendocrinology21263307. (doi:10.1006/frne.2000.0197)

    • Search Google Scholar
    • Export Citation
  • AhrenB2000Diurnal variation in circulating leptin is dependent on gender, food intake and circulating insulin in mice. Acta Physiologica Scandinavica169325331. (doi:10.1046/j.1365-201x.2000.00746.x)

    • Search Google Scholar
    • Export Citation
  • BartellSMRayalamSAmbatiSGaddamDRHartzellDLHamrickMSheJXDella-FeraMABaileCA2011Central (ICV) leptin injection increases bone formation, bone mineral density, muscle mass, serum IGF-1, and the expression of osteogenic genes in leptin-deficient ob/ob mice. Journal of Bone and Mineral Research2617101720. (doi:10.1002/jbmr.406)

    • Search Google Scholar
    • Export Citation
  • BaumTYapSPKarampinosDCNardoLKuoDBurghardtAJMasharaniUBSchwartzAVLiXLinkTM2012Does vertebral bone marrow fat content correlate with abdominal adipose tissue, lumbar spine bone mineral density, and blood biomarkers in women with type 2 diabetes mellitus?Journal of Magnetic Resonance Imaging35117124. (doi:10.1002/jmri.22757)

    • Search Google Scholar
    • Export Citation
  • BiancoPRobeyPGSimmonsPJ2008Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell2313319. (doi:10.1016/j.stem.2008.03.002)

    • Search Google Scholar
    • Export Citation
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